Dry powder compositions of treprostinil prodrugs and methods of use thereof

ABSTRACT

or an enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof, (b) from about 0.01 wt % to about 3 wt % of DSPE-PEG2000, (c) from about 10 wt % to about 50 wt % of leucine, and the balance being (d) a sugar selected from the group consisting of trehalose and mannitol. The entirety of (a), (b), (c), and (d) is 100 wt %, and R1 is tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl. The method includes administering an effective amount of the dry powder composition to the lungs of the patient by inhalation via a dry powder inhaler. In certain compositions and methods provided herein, R1 is hexadecyl, e.g., linear hexadecyl.

CROSS REFERENCE T0 RELATED APPLICATION

This application claims priority from U.S. Provisional Application Ser.No. 62/840,186, filed Apr. 29, 2019, the disclosure of which isincorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Pulmonary hypertension (PH) is characterized by an abnormally high bloodpressure in the lung vasculature. It is a progressive, lethal diseasethat leads to heart failure and can occur in the pulmonary artery,pulmonary vein, or pulmonary capillaries. Symptomatically patientsexperience shortness of breath, dizziness, fainting, and other symptoms,all of which are made worse by exertion. There are multiple causes, andcan be of unknown origin, idiopathic, and can lead to hypertension inother systems, for example, portopulmonary hypertension in whichpatients have both portal and pulmonary hypertension.

Pulmonary hypertension has been classified into five groups by the WorldHealth Organization (WHO). Group 1 is called pulmonary arterialhypertension (PAH), and includes PAH that has no known cause(idiopathic), inherited PAH (i.e., familial PAH or FPAH), PAH that iscaused by drugs or toxins, and PAH caused by conditions such asconnective tissue diseases, HIV infection, liver disease, and congenitalheart disease. Group 2 pulmonary hypertension is characterized aspulmonary hypertension associated with left heart disease. Group 3pulmonary hypertension is characterized as PH associated with lungdiseases, such as chronic obstructive pulmonary disease and interstitiallung diseases, as well as PH associated with sleep-related breathingdisorders (e.g., sleep apnea). Group 4 PH is PH due to chronicthrombotic and/or embolic disease, e.g., PH caused by blood clots in thelungs or blood clotting disorders. Group 5 includes PH caused by otherdisorders or conditions, e.g., blood disorders (e.g., polycythemia vera,essential thrombocythemia), systemic disorders (e.g., sarcoidosis,vasculitis), and metabolic disorders (e.g., thyroid disease, glycogenstorage disease).

Pulmonary arterial hypertension (PAH) afflicts approximately 200,000people globally with approximately 30,000-40,000 of those patients inthe United States. PAH patients experience constriction of pulmonaryarteries which leads to high pulmonary arterial pressures, making itdifficult for the heart to pump blood to the lungs. Patients suffer fromshortness of breath and fatigue which often severely limits the abilityto perform physical activity.

The New York Heart Association (NYHA) has categorized PAH patients intofour functional classes to rate the severity of the disease. Class I PAHpatients as categorized by the NYHA do not have a limitation of physicalactivity, as ordinary physical activity does not cause undue dyspnoea orfatigue, chest pain, or near syncope. Class II PAH patients ascategorized by the NYHA have a slight limitation on physical activity.These patients are comfortable at rest, but ordinary physical activitycauses undue dyspnoea or fatigue, chest pain or near syncope. Class IIIPAH patients as categorized by the NYHA have a marked limitation ofphysical activity. Although comfortable at rest, class III PAH patientsexperience undue dyspnoea or fatigue, chest pain or near syncope as aresult of less than ordinary physical activity. Class IV PAH patients ascategorized by the NYHA are unable to carry out any physical activitywithout symptoms. Class IV PAH patients might experience dyspnoea and/orfatigue at rest, and discomfort is increased by any physical activity.Signs of right heart failure are often manifested by class IV PAHpatients.

Patients with PAH are treated with an endothelin receptor antagonist(ERA), phosphodiesterase type 5 (PDE-5) inhibitor, a guanylate cyclasestimulator, a prostanoid (e.g., prostacyclin), or a combination thereof.ERAs include abrisentan (Letairis®), sitaxentan, bosentan (Tracleer®),and macitentan (Opsumit®). PDE-5 inhibitors indicated for the treatmentof PAH include sildenafil (Revatio®) and tadalafil (Adcirca®).Prostanoids indicated for the treatment of PAH include iloprost,epoprosentol and treprostinil (Remodulin®, Tyvaso®). The one approvedguanylate cyclase stimulator is riociguat (Adempas®). Additionally,patients are often treated with combinations of the aforementionedcompounds.

Portopulmonary hypertension (PPH) is defined by the coexistence ofportal and pulmonary hypertension, and is a serious complication ofliver disease. The diagnosis of portopulmonary hypertension is based onhemodynamic criteria: (1) portal hypertension and/or liver disease(clinical diagnosis-ascites/varices/splenomegaly), (2) mean pulmonaryartery pressure >25 mmHg at rest, (3) pulmonary vascular resistance >240dynes s/cm⁵, (4) pulmonary artery occlusion pressure <15 mmHg ortranspulmonary gradient >12 mmHg. PPH is a serious complication of liverdisease, and is present in 0.25 to 4% of patients suffering fromcirrhosis. Today, PPH is comorbid in 4-6% of those referred for a livertransplant.

Pulmonary fibrosis is a respiratory disease in which scars are formed inthe lung tissues, leading to serious breathing problems. Scar formation,i.e., the accumulation of excess fibrous connective tissue, leads tothickening of the walls, and causes reduced oxygen supply in the blood.As a result, pulmonary fibrosis patients suffer from perpetual shortnessof breath. In some patients the specific cause of the disease can bediagnosed, but in others the probable cause cannot be determined, acondition called idiopathic pulmonary fibrosis.

The present invention addresses the need for novel treatment options forpulmonary hypertension (PH) (including pulmonary arterial hypertension(PAH)), portopulmonary hypertension (PPH), and pulmonary fibrosis byproviding dry powder compositions of treprostinil prodrugs useful forpulmonary administration, and methods for administering the same topatients in need of treatment.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure relates to a dry powdercomposition. The dry powder composition includes (a) from about 0.1 wt %to about 3 wt % of a compound of Formula (I):

or an enantiomer, diastereomer, or a pharmaceutically acceptable saltthereof, wherein R¹ is tetradecyl, pentadecyl, hexadecyl, heptadecyl, oroctadecyl; (b) from about 0.01 wt % to about 3 wt % of distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), (c) from about 10 wt % to about 50 wt % of leucine, andthe balance being (d) a sugar selected from the group consisting oftrehalose and mannitol. The entirety of (a), (b), (c), and (d) is 100 wt%.

In one embodiment, R¹ is tetradecyl. In a further embodiment, R¹ islinear tetradecyl.

In one embodiment, R¹ is pentadecyl. In a further embodiment, R¹ islinear pentadecyl.

In one embodiment, R¹ is heptadecyl. In a further embodiment, R¹ islinear heptadecyl.

In one embodiment, R¹ is octadecyl. In a further embodiment, R¹ islinear octadecyl.

In one embodiment, R¹ is hexadecyl. In a further embodiment, R¹ islinear hexadecyl.

In one embodiment, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, is presentat from about 0.5 wt % to about 2 wt % of the total weight of the drypowder composition, and the DSPE-PEG2000 is present at from about 0.05wt % to about 2 wt % of the total weight of the dry powder composition.In a further embodiment, R¹ is hexadecyl. In even a further embodiment,R¹ is linear hexadecyl. In a further embodiment, the DSPE-PEG2000 ispresent at from about 0.15 wt % to about 1.4 wt % of the total weight ofthe dry powder composition. In even a further embodiment, theDSPE-PEG2000 is present at from about 0.25 wt % to about 1 wt % of thetotal weight of the dry powder composition.

In one embodiment, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat from about 1 wt % to about 2 wt % of the total weight of the drypowder composition, and the DSPE-PEG2000 is present at from about 0.1 wt% to about 2 wt % of the total weight of the dry powder composition. Ina further embodiment, R¹ is hexadecyl. In even a further embodiment, R¹is linear hexadecyl. In a further embodiment, the DSPE-PEG2000 ispresent at from about 0.3 wt % to about 1.4 wt % of the total weight ofthe dry powder composition. In even a further embodiment, theDSPE-PEG2000 is present at from about 0.5 wt % to about 1 wt % of thetotal weight of the dry powder composition.

In one embodiment, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat from about 1.2 wt % to about 1.8 wt % of the total weight of the drypowder composition, and the DSPE-PEG2000 is present at from about 0.12wt % to about 1.8 wt % of the total weight of the dry powdercomposition. In a further embodiment, R¹ is hexadecyl. In even a furtherembodiment, R¹ is linear hexadecyl. In a further embodiment, theDSPE-PEG2000 is present at from about 0.36 wt % to about 1.26 wt % ofthe total weight of the dry powder composition. In even a furtherembodiment, the DSPE-PEG2000 is present at from about 0.6 wt % to about0.9 wt % of the total weight of the dry powder composition.

In one embodiment, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat from about 1 wt % to about 1.5 wt % of the total weight of the drypowder composition, and the DSPE-PEG2000 is present at from about 0.1 wt% to about 1.5 wt % of the total weight of the dry powder composition.In a further embodiment, R¹ is hexadecyl. In even a further embodiment,R¹ is linear hexadecyl. In a further embodiment, the DSPE-PEG2000 ispresent at from about 0.3 wt % to about 1.05 wt % of the total weight ofthe dry powder composition. In even a further embodiment, theDSPE-PEG2000 is present at from about 0.5 wt % to about 0.75 wt % of thetotal weight of the dry powder composition.

In one embodiment, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat from about 1.4 wt % to about 1.6 wt % of the total weight of the drypowder composition, and the DSPE-PEG2000 is present at from about 0.14wt % to about 1.6 wt % of the total weight of the dry powdercomposition. In a further embodiment, R¹ is hexadecyl. In even a furtherembodiment, R¹ is linear hexadecyl. In a further embodiment, theDSPE-PEG2000 is present at from about 0.42 wt % to about 1.12 wt % ofthe total weight of the dry powder composition. In even a furtherembodiment, the DSPE-PEG2000 is present at from about 0.7 wt % to about0.8 wt % of the total weight of the dry powder composition.

In one embodiment, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat about 1.5 wt % of the total weight of the dry powder composition, andthe DSPE-PEG2000 is present at from about 0.15 wt % to about 1.5 wt % ofthe total weight of the dry powder composition. In a further embodiment,R¹ is hexadecyl. In even a further embodiment, R¹ is linear hexadecyl.In a further embodiment, the DSPE-PEG2000 is present at from about 0.45wt % to about 1.05 wt % of the total weight of the dry powdercomposition. In even a further embodiment, the DSPE-PEG2000 is presentat about 0.75 wt % of the total weight of the dry powder composition.

In one embodiment, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat from about 0.1 wt % to about 3 wt % of the total weight of the drypowder composition, and the weight ratio of the DSPE-PEG2000 to thecompound of Formula (I), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof is in a range of from about0.1:1 (DSPE-PEG2000: the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof) to about1:1 (DSPE-PEG2000: the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof). In afurther embodiment, R¹ is hexadecyl. In even a further embodiment, R¹ islinear hexadecyl. In a further embodiment, the compound of Formula (I),or an enantiomer, diastereomer, or a pharmaceutically acceptable saltthereof is present at from about 0.5 wt % to about 2 wt % of the totalweight of the dry powder composition. In a further embodiment, thecompound of Formula (I), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof is present at from about 1 wt %to about 2 wt % of the total weight of the dry powder composition. In afurther embodiment, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat from about 1.2 wt % to about 1.8 wt % of the total weight of the drypowder composition. In a further embodiment, the compound of Formula(I), or an enantiomer, diastereomer, or a pharmaceutically acceptablesalt thereof is present at from about 1 wt % to about 1.5 wt % of thetotal weight of the dry powder composition. In a further embodiment, thecompound of Formula (I), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof is present at from about 1.4 wt% to about 1.6 wt % of the total weight of the dry powder composition.In even a further embodiment, the compound of Formula (I), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at about 1.5 wt % of the total weight of the dry powdercomposition. In another embodiment, the weight ratio of the DSPE-PEG2000to the compound of Formula (I), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof is in a range of from about0.3:1 (DSPE-PEG2000: the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof) to about0.7:1 (DSPE-PEG2000: the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof). In afurther embodiment, the weight ratio of the DSPE-PEG2000 to the compoundof Formula (I), or an enantiomer, diastereomer, or a pharmaceuticallyacceptable salt thereof is about 0.5:1 (DSPE-PEG2000: the compound ofFormula (I), or an enantiomer, diastereomer, or a pharmaceuticallyacceptable salt thereof).

In one embodiment of a dry powder composition provided herein, theleucine is present at from about 15 wt % to about 40 wt % of the totalweight of the dry powder composition. In a further embodiment, R¹ ishexadecyl. In even a further embodiment, R¹ is linear hexadecyl. Inanother embodiment, the leucine is present at from about 18 wt % toabout 33 wt % of the total weight of the dry powder composition. In afurther embodiment, R¹ is hexadecyl. In even a further embodiment, R¹ islinear hexadecyl. In another embodiment, the leucine is present at fromabout 20 wt % to about 30 wt % of the total weight of the dry powdercomposition. In a further embodiment, R¹ is hexadecyl. In even a furtherembodiment, R¹ is linear hexadecyl. In another embodiment, the leucineis present at from about 25 wt % to about 30 wt % of the total weight ofthe dry powder composition. In a further embodiment, R¹ is hexadecyl. Ineven a further embodiment, R¹ is linear hexadecyl. In anotherembodiment, the leucine is present at from about 27 wt % to about 30 wt% of the total weight of the dry powder composition. In a furtherembodiment, R¹ is hexadecyl. In even a further embodiment, R¹ is linearhexadecyl. In another embodiment, the leucine is present at about 30 wt% of the total weight of the dry powder composition. In a furtherembodiment, R¹ is hexadecyl. In even a further embodiment, R¹ is linearhexadecyl.

In one embodiment, the sugar is mannitol. In a further embodiment, R¹ ishexadecyl. In a further embodiment, R¹ is linear hexadecyl.

In one embodiment, the dry powder composition includes (a) about 1.5 wt% of the compound of Formula (I), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof, (b) about 0.7 wt % of theDSPE-PEG2000, (c) about 29.3 wt % of the leucine, and the balance being(d) mannitol. In a further embodiment, R¹ is hexadecyl. In a furtherembodiment, R¹ is linear hexadecyl.

In one embodiment, the dry powder composition includes (a) about 1.5 wt% of the compound of Formula (I), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof, (b) about 0.75 wt % of theDSPE-PEG2000, (c) about 29.30 wt % of the leucine, and (d) about 68.45wt % of the mannitol. In a further embodiment, R¹ is hexadecyl. In afurther embodiment, R¹ is linear hexadecyl.

In one embodiment, the dry powder composition is in the form of anaerosol having particles with a mass median aerodynamic diameter (MMAD)of from about 1 μm to about 3 as measured by Next Generation Impactor(NGI). In a further embodiment, the dry powder composition is in theform of an aerosol having particles with an MMAD of from about 1.3 μm toabout 2.0 as measured by NGI. In a further embodiment, R¹ is hexadecyl.In a further embodiment, R¹ is linear hexadecyl.

In one embodiment, the sugar is mannitol, and the dry powder compositionis in the form of an aerosol having particles with an MMAD of from about1 μm to about 3 μm, as measured by NGI. In another embodiment, the sugaris mannitol, and the dry powder composition is in the form of an aerosolhaving particles with an MMAD of from about 1.7 μm to about 2.7 μm, asmeasured by NGI. In a further embodiment, R¹ is hexadecyl. In a furtherembodiment, R¹ is linear hexadecyl.

In one embodiment, the dry powder composition is in the form of anaerosol having particles with a fine particle fraction (FPF) of fromabout 30% to about 60%, as measured by NGI. In a further embodiment, R¹is hexadecyl. In a further embodiment, R¹ is linear hexadecyl.

In another aspect, the present disclosure relates to a method fortreating pulmonary hypertension in a patient in need thereof. The methodincludes administering an effective amount of the dry powder compositiondisclosed herein to the lungs of the patient by inhalation via a drypowder inhaler.

The pulmonary hypertension, in one embodiment, is pulmonary arterialhypertension (PAH). The PAH, in one embodiment, is class I PAH, ascharacterized by the New York Heart Association (NYHA). In anotherembodiment, the PAH is class II PAH, as characterized by NYHA. Inanother embodiment, the PAH is class III PAH, as characterized by NYHA.In another embodiment, the PAH is class IV PAH, as characterized byNYHA.

In one embodiment, the pulmonary hypertension is group 1 pulmonaryhypertension, as characterized by the World Health Organization (WHO).

In another embodiment, the pulmonary hypertension is group 2 pulmonaryhypertension, as characterized by the WHO.

In another embodiment, the pulmonary hypertension is group 3 pulmonaryhypertension, as characterized by the WHO.

In another embodiment, the pulmonary hypertension is group 4 pulmonaryhypertension, as characterized by the WHO.

In another embodiment, the pulmonary hypertension is group 5 pulmonaryhypertension, as characterized by the WHO.

In still another aspect, the present disclosure relates to a method fortreating portopulmonary hypertension or pulmonary fibrosis in a patientin need thereof. The method includes administering an effective amountof the dry powder composition disclosed herein to the lungs of thepatient by inhalation via a dry powder inhaler.

In one embodiment of the treatment methods described herein, theadministering is conducted in a once-a-day, twice-a-day, orthree-times-a-day dosing.

In another embodiment of the treatment methods described herein, theadministering includes aerosolizing the dry powder composition andadministering an aerosolized dry powder composition to the lungs of thepatient via inhalation. In one embodiment, the aerosolized dry powdercomposition includes particles with an MMAD of from about 1 μm to about3 μm, as measured by NGI. In another embodiment, the aerosolized drypowder composition includes particles with an FPF of from about 30% toabout 60%, as measured by NGI.

In still another aspect, the present disclosure relates to a system fortreating pulmonary hypertension, portopulmonary hypertension, orpulmonary fibrosis. The system includes one of the dry powdercompositions disclosed herein and a dry powder inhaler (DPI).

The DPI, in one embodiment, is either a single dose or a multidoseinhaler.

In another embodiment, the DPI is pre-metered or device-metered.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the effect of leucine content on spray dryingrecovery of mannitol-based C16TR (treprostinil palmitil) dry powders.

FIGS. 2A-2D are SEM images of mannitol-based C16TR (treprostinilpalmitil) dry powders containing different amounts of leucine asindicated.

FIG. 3 is a graph showing the effect of leucine content on particle sizedistribution measured by laser diffraction in mannitol-based C16TR(treprostinil palmitil) dry powders.

FIG. 4 is a graph showing the effect of C16TR (treprostinil palmitil)content on MMAD of mannitol-based C16TR (treprostinil palmitil) drypowders.

FIGS. 5A-5C are SEM images of mannitol-based C16TR (treprostinilpalmitil) dry powders spray dried at different inlet temperatures. Theimages of the upper panel were taken at high magnification and theimages of the lower panel were taken at low magnification.

FIGS. 6A and 6B are SEM images showing the morphology of mannitol-basedC16TR (treprostinil palmitil) dry powders spray dried at the inlettemperature of 135° C. with or without ammonium bicarbonate (ABC, 0.5mg/mL). The images of the upper panel were taken at high magnificationand the images of the lower panel were taken at low magnification.

FIG. 7A is a graph showing the DSC data of the mannitol-based C16TR(treprostinil palmitil) dry powder batch SD-NNP-179.

FIG. 7B is a graph showing the X-ray diffraction data of themannitol-based C16TR (treprostinil palmitil) dry powder batchSD-NNP-179.

FIGS. 8A and 8B are SEM images showing the effect of leucine content onthe morphology of the trehalose-based C16TR (treprostinil palmitil) drypowders.

FIGS. 9A-9C are SEM images showing the effect of spray drying inlettemperature on the morphology of the trehalose-based C16TR (treprostinilpalmitil) dry powders.

FIG. 10A is a graph showing the DSC data of the trehalose-based C16TR(treprostinil palmitil) dry powders.

FIG. 10B is a graph showing the X-ray diffraction data of thetrehalose-based C16TR (treprostinil palmitil) dry powders.

FIG. 11 is a DVS isotherm plot showing moisture absorption of themannitol-based C16TR (treprostinil palmitil) dry powder batch SD-NNP-167(C16TR (treprostinil palmitil)/DSPE-PEG2000/Man/Leu, 1/0.5/80/20).

FIG. 12 is a DVS isotherm plot showing moisture absorption of thetrehalose-based C16TR (treprostinil palmitil) dry powder batchSD-NNP-162 (C16TR (treprostinil palmitil)/DSPE-PEG2000/Treh/Leu,1/0.5/80/20).

FIG. 13 is a DVS isotherm plot showing moisture absorption of thetrehalose-based C16TR (treprostinil palmitil) dry powder batchSD-NNP-163 (C16TR (treprostinil palmitil)/DSPE-PEG2000/Treh/Leu,1/0.5/70/30).

FIG. 14 is a graph showing the changes in MMAD in an acceleratedstability study of the mannitol-based C16TR (treprostinil palmitil) drypowders.

FIGS. 15A and 15B are SEM images of the mannitol-based C16TR(treprostinil palmitil) dry powder batch SD-NNP-179 with 1% of C16TR(treprostinil palmitil) at T0 and T3 (3 months), respectively.

FIGS. 16A and 16B are SEM images of the mannitol-based C16TR(treprostinil palmitil) dry powder batch SD-NNP-183 with 1.5% of C16TR(treprostinil palmitil) at T0 and T3 (3 months), respectively.

FIGS. 17A and 17B are SEM images of the mannitol-based C16TR(treprostinil palmitil) dry powder batch SD-NNP-184 with 2% of C16TR(treprostinil palmitil) at T0 and T3 (3 months), respectively.

FIGS. 18A and 18B are SEM images of the mannitol-based C16TR(treprostinil palmitil) dry powder batches with 3% of C16TR(treprostinil palmitil) (SD-NNP-190) and 5% of C16TR (treprostinilpalmitil) (SD-NNP-191), respectively, at T5 (5 months).

FIG. 19 is a graph showing the changes in MMAD in an acceleratedstability study of the trehalose-based C16TR (treprostinil palmitil) drypowders.

FIG. 20 is a graph showing the changes in FPF in an acceleratedstability study of the trehalose-based C16TR (treprostinil palmitil) drypowders.

FIGS. 21A and 21B are SEM images of the trehalose-based C16TR(treprostinil palmitil) dry powder batch SD-NNP-162 with 1% of C16TR(treprostinil palmitil) at T0 and T3.5 (3.5 months), respectively.

FIGS. 22A and 22B are SEM images of the trehalose-based C16TR(treprostinil palmitil) dry powder batch SD-NNP-163 with 1% of C16TR(treprostinil palmitil) at T0 and T3.5 (3.5 months), respectively.

FIGS. 23A and 23B are SEM images of the trehalose-based C16TR(treprostinil palmitil) dry powder batch SD-NNP-188 with 1.5% of C16TR(treprostinil palmitil) at T0 and T3.5 (3.5 months), respectively.

FIGS. 24A and 24B are SEM images of the trehalose-based C16TR(treprostinil palmitil) dry powder batch SD-NNP-189 with 2% of C16TR(treprostinil palmitil) at T0 and T3.5 (3.5 months), respectively.

FIG. 25 is a graph showing pressure titration of spray driedtreprostinil palmitil dry powder formulations A, B, C, and D. Forvisibility, data points are offset in order (A, B, C and D), within eachair pressure category (left to right).

FIG. 26 is a graph showing particle size distributions of treprostinilpalmitil dry powder formulations A, B, C, and D.

FIG. 27 is an SEM image of treprostinil palmitil dry powder formulationA.

FIG. 28 is an SEM image of treprostinil palmitil dry powder formulationB.

FIG. 29 is an SEM image of treprostinil palmitil dry powder formulationC.

FIG. 30 is an SEM image of treprostinil palmitil dry powder formulationD.

FIG. 31 is a graph showing the fine particle doses (FPD) of treprostinilpalmitil dry powder formulation A at T=0 and after stored in capsulesfor 1-3 months at 25° C. or 40° C. as indicated, or after stored as bulkfor 3 months at 25° C. or 40° C. and filled into capsules and dosed onthe same day.

FIG. 32 is a graph showing the fine particle doses (FPD) of treprostinilpalmitil dry powder formulation C at T=0 and after stored in capsulesfor 1-3 months at 25° C. or 40° C. as indicated, or after stored as bulkfor 3 months at 25° C. or 40° C. and filled into capsules and dosed onthe same day.

FIG. 33 is a graph showing the fine particle doses (FPD) of treprostinilpalmitil dry powder formulation D at T=0 and after stored in capsulesfor 1-3 months at 25° C. or 40° C. as indicated, or after stored as bulkfor 3 months at 25° C. or 40° C. and filled into capsules and dosed onthe same day.

FIG. 34 is a dynamic vapor sorption (DVS) isotherm plot of thetrehalose-based C16TR (treprostinil palmitil) dry powder formulation.

FIG. 35 is a graph showing the aerosol particle size distribution of thetrehalose-based C16TR (treprostinil palmitil) dry powder formulation atT=0 month, and after stored at 40° C. and uncontrolled ambient humidityfor 1.5 months, 2.5 months, and 3.5 months (n=1 per time point).

FIG. 36 is a graph showing the concentration of C16TR (treprostinilpalmitil) equivalent (C16TR (treprostinil palmitil) plus treprostinil,ng/g) in the lung after inhalation of the trehalose-based C16TR(treprostinil palmitil) dry powder formulation and nebulized INS1009.

FIG. 37 is a graph showing the concentration of C16TR (treprostinilpalmitil) equivalent (C16TReq) in the lungs after inhaled treprostinilpalmitil dry powder formulation D or formulation C.

FIG. 38 is a graph showing the concentration of C16TR (treprostinilpalmitil) in the lungs after inhaled treprostinil palmitil dry powderformulation D or formulation C.

FIG. 39 is a graph showing the concentration of TRE in the lungs afterinhaled treprostinil palmitil dry powder formulation D or formulation C.

FIG. 40 is a graph showing the concentration of TRE in the plasma afterinhaled treprostinil palmitil dry powder formulation D or formulation C.

FIG. 41 is a graph showing the ΔRVPP response to hypoxic challenge inrats exposed to treprostinil palmitil dry powder formulation D orformulation C.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present disclosure, the term “about” may be used inconjunction with numerical values and/or ranges. The term “about” isunderstood to mean those values near to a recited value. For example,“about 40 [units]” may mean within ±25% of 40 (e.g., from 30 to 50),within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%,less than ±1%, or any other value or range of values therein or therebelow.

The term “pharmaceutically acceptable salt” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids includinginorganic or organic bases and inorganic or organic acids. The nature ofthe salt is not critical, provided that it is pharmaceuticallyacceptable. Suitable pharmaceutically acceptable acid addition salts maybe prepared from an inorganic acid or from an organic acid. Exemplarypharmaceutical salts are disclosed in Stahl, P. H., Wermuth, C. G., Eds.Handbook of Pharmaceutical Salts: Properties, Selection and Use; VerlagHelvetica Chimica Acta/Wiley-VCH: Zurich, 2002, the contents of whichare hereby incorporated by reference in their entirety. Specificnon-limiting examples of inorganic acids are hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriateorganic acids include, without limitation, aliphatic, cycloaliphatic,aromatic, arylaliphatic, and heterocyclyl containing carboxylic acidsand sulfonic acids, for example formic, acetic, propionic, succinic,glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic,anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic,phenylacetic, mandelic, embonic (pamoic), methanesulfonic,ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic,2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic,3-hydroxybutyric, galactaric or galacturonic acid. Suitablepharmaceutically acceptable salts of free acid-containing compoundsdisclosed herein include, without limitation, metallic salts and organicsalts. Exemplary metallic salts include, but are not limited to,appropriate alkali metal (group Ia) salts, alkaline earth metal (groupIIa) salts, and other physiological acceptable metals. Such salts can bemade from aluminum, calcium, lithium, magnesium, potassium, sodium andzinc. Exemplary organic salts can be made from primary amines, secondaryamines, tertiary amines and quaternary ammonium salts, for example,tromethamine, diethylamine, tetra-N-methylammonium,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine.

Throughout the present specification, numerical ranges are provided forcertain quantities. It is to be understood that these ranges compriseall subranges therein. Thus, the range “50-80” includes all possibleranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.).Furthermore, all values within a given range may be an endpoint for therange encompassed thereby (e.g., the range 50-80 includes the rangeswith endpoints such as 55-80, 50-75, etc.).

The term “treating” in one embodiment, includes: (1) preventing ordelaying the appearance of clinical symptoms of the state, disorder orcondition developing in the subject that may be afflicted with orpredisposed to the state, disorder or condition but does not yetexperience or display clinical or subclinical symptoms of the state,disorder or condition; (2) inhibiting the state, disorder or condition(e.g., arresting, reducing or delaying the development of the disease,or a relapse thereof in case of maintenance treatment, of at least oneclinical or subclinical symptom thereof); and/or (3) relieving thecondition (e.g., causing regression of the state, disorder or conditionor at least one of its clinical or subclinical symptoms). In oneembodiment, “treating” refers to inhibiting the state, disorder orcondition (e.g., arresting, reducing or delaying the development of thedisease, or a relapse thereof in case of maintenance treatment, of atleast one clinical or subclinical symptom thereof). In anotherembodiment, “treating” refers to relieving the condition (for example,by causing regression of the state, disorder or condition or at leastone of its clinical or subclinical symptoms). The benefit to a subjectto be treated is either statistically significant as compared to thestate or condition of the same subject before the treatment, or ascompared to the state or condition of an untreated control subject, orthe benefit is at least perceptible to the subject or to the physician.

“Effective amount” means an amount of a dry powder composition of thepresent disclosure that is sufficient to result in the desiredtherapeutic response.

In one aspect of the present invention, a dry powder composition of atreprostinil prodrug is provided. The dry powder composition includes:

(a) a compound of Formula (I):

or an enantiomer, diastereomer, or a pharmaceutically acceptable saltthereof, wherein R¹ is tetradecyl, pentadecyl, hexadecyl, heptadecyl, oroctadecyl, and the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat from about 0.1 wt % to about 3 wt % of the total weight of the drypowder composition;

(b) from about 0.01 wt % to about 3 wt % of DSPE-PEG2000,

(c) from about 10 wt % to about 50 wt % of leucine, and the balancebeing

(d) a sugar selected from the group consisting of trehalose andmannitol. The entirety of (a), (b), (c), and (d) is 100 wt %.

In some embodiments, the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat about 0.1 wt %, about 0.3 wt %, about 0.5 wt %, about 0.7 wt %, about1 wt %, about 1.3 wt %, about 1.5 wt %, about 1.7 wt %, about 2.0 wt %,about 2.3 wt %, about 2.5 wt %, about 2.7 wt %, or about 3 wt % of thetotal weight of the dry powder composition. In a further embodiment, thecompound of Formula (I), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof is present at about 1.5 wt % ofthe total weight of the dry powder composition. The compound of Formula(I) and pharmaceutically acceptable salts thereof are treprostinilprodrugs as disclosed in International Application Publication WO2015/061720, the disclosure of which is incorporated herein by referencein its entirety. In some embodiments, the leucine is present at about 10wt %, about 15 wt %, about 20 wt %, about 25 wt %, about 30 wt %, about35 wt %, about 40 wt %, about 45 wt %, or about 50 wt % of the totalweight of the dry powder composition.

PEG refers to polyethylene glycol, also known as polyethylene oxide(PEO) or polyoxyethylene (POE), depending on its molecular weight. TheDSPE-PEG2000 may include a branched or unbranched PEG molecule with anaverage PEG molecular weight of 2000 g/mol. In one embodiment, (b) isDSPE-PEG2000 present at from about 0.03 wt % to about 2.1 wt % of thetotal weight of the dry powder composition. In another embodiment, (b)is DSPE-PEG2000 present at from about 0.05 wt % to about 1.5 wt % of thetotal weight of the dry powder composition.

In one embodiment of the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, R¹ istetradecyl. In a further embodiment, R¹ is linear tetradecyl.

In another embodiment of the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, R¹ ispentadecyl. In a further embodiment, R¹ is linear pentadecyl.

In another embodiment of the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, R¹ isheptadecyl. In a further embodiment, R¹ is linear heptadecyl.

In another embodiment of the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, R¹ isoctadecyl. In a further embodiment, R¹ is linear octadecyl.

In another embodiment of the compound of Formula (I), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, R¹ ishexadecyl. In a further embodiment, R¹ is linear hexadecyl, i.e., thecompound of Formula (I), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof, is a compound of Formula (II):

or an enantiomer, diastereomer, or a pharmaceutically acceptable saltthereof. In one embodiment, the compound of Formula (I) or apharmaceutically acceptable salt thereof is a compound of Formula (II)or a pharmaceutically acceptable salt thereof. In a further embodiment,the compound of Formula (I) or a pharmaceutically acceptable saltthereof is a compound of Formula (II). In a further embodiment, thecompound of Formula (I) is a compound of Formula (II). The compound ofFormula (II) is also referred to herein as C16TR or treprostinilpalmitil. In the present application, C16TR and treprostinil palmitilare used interchangeably and refer to the compound of Formula (II).

In one embodiment, (a) is a compound of Formula (I) or apharmaceutically acceptable salt thereof. In a further embodiment, (a)is a compound of Formula (I). In another embodiment, (a) is a compoundof Formula (II) or a pharmaceutically acceptable salt thereof. In afurther embodiment, (a) is a compound of Formula (II).

In one embodiment, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at from about 0.5 wt % to about 2 wt % of the total weight ofthe dry powder composition, and the DSPE-PEG2000 is present at fromabout 0.05 wt % to about 2 wt % of the total weight of the dry powdercomposition. In a further embodiment, the DSPE-PEG2000 is present atfrom about 0.15 wt % to about 1.4 wt % of the total weight of the drypowder composition. In a further embodiment, the DSPE-PEG2000 is presentat from about 0.25 wt % to about 1 wt % of the total weight of the drypowder composition. In some embodiments, the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof is present at fromabout 0.5 wt % to about 2 wt % of the total weight of the dry powdercomposition. In some embodiments, the compound of Formula (I) or (II) ispresent at from about 0.5 wt % to about 2 wt % of the total weight ofthe dry powder composition.

In one embodiment, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at from about 1 wt % to about 2 wt % of the total weight ofthe dry powder composition, and the DSPE-PEG2000 is present at fromabout 0.1 wt % to about 2 wt % of the total weight of the dry powdercomposition. In a further embodiment, the DSPE-PEG2000 is present atfrom about 0.3 wt % to about 1.4 wt % of the total weight of the drypowder composition. In a further embodiment, the DSPE-PEG2000 is presentat from about 0.5 wt % to about 1 wt % of the total weight of the drypowder composition. In some embodiments, the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof is present at fromabout 1 wt % to about 2 wt % of the total weight of the dry powdercomposition. In some embodiments, the compound of Formula (I) or (II) ispresent at from about 1 wt % to about 2 wt % of the total weight of thedry powder composition.

In one embodiment, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at from about 1.2 wt % to about 1.8 wt % of the total weightof the dry powder composition, and the DSPE-PEG2000 is present at fromabout 0.12 wt % to about 1.8 wt % of the total weight of the dry powdercomposition. In a further embodiment, the DSPE-PEG2000 is present atfrom about 0.36 wt % to about 1.26 wt % of the total weight of the drypowder composition. In a further embodiment, the DSPE-PEG2000 is presentat from about 0.6 wt % to about 0.9 wt % of the total weight of the drypowder composition. In some embodiments, the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof is present at fromabout 1.2 wt % to about 1.8 wt % of the total weight of the dry powdercomposition. In some embodiments, the compound of Formula (I) or (II) ispresent at from about 1.2 wt % to about 1.8 wt % of the total weight ofthe dry powder composition.

In one embodiment, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at from about 1 wt % to about 1.5 wt % of the total weight ofthe dry powder composition, and the DSPE-PEG2000 is present at fromabout 0.1 wt % to about 1.5 wt % of the total weight of the dry powdercomposition. In a further embodiment, the DSPE-PEG2000 is present atfrom about 0.3 wt % to about 1.05 wt % of the total weight of the drypowder composition. In a further embodiment, the DSPE-PEG2000 is presentat from about 0.5 wt % to about 0.75 wt % of the total weight of the drypowder composition. In some embodiments, the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof is present at fromabout 1 wt % to about 1.5 wt % of the total weight of the dry powdercomposition. In some embodiments, the compound of Formula (I) or (II) ispresent at from about 1 wt % to about 1.5 wt % of the total weight ofthe dry powder composition.

In one embodiment, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at from about 1.4 wt % to about 1.6 wt % of the total weightof the dry powder composition, and the DSPE-PEG2000 is present at fromabout 0.14 wt % to about 1.6 wt % of the total weight of the dry powdercomposition. In a further embodiment, the DSPE-PEG2000 is present atfrom about 0.42 wt % to about 1.12 wt % of the total weight of the drypowder composition. In a further embodiment, the DSPE-PEG2000 is presentat from about 0.7 wt % to about 0.8 wt % of the total weight of the drypowder composition. In some embodiments, the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof is present at fromabout 1.4 wt % to about 1.6 wt % of the total weight of the dry powdercomposition. In some embodiments, the compound of Formula (I) or (II) ispresent at from about 1.4 wt % to about 1.6 wt % of the total weight ofthe dry powder composition.

In one embodiment, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at about 1 wt % of the total weight of the dry powdercomposition, and the DSPE-PEG2000 is present at from about 0.1 wt % toabout 1 wt % of the total weight of the dry powder composition. In afurther embodiment, the DSPE-PEG2000 is present at from about 0.3 wt %to about 0.7 wt % of the total weight of the dry powder composition. Ina further embodiment, the DSPE-PEG2000 is present at about 0.5 wt % ofthe total weight of the dry powder composition. In some embodiments, thecompound of Formula (I) or (II), or a pharmaceutically acceptable saltthereof is present at about 1 wt % of the total weight of the dry powdercomposition. In some embodiments, the compound of Formula (I) or (II) ispresent at about 1 wt % of the total weight of the dry powdercomposition.

In one embodiment, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at about 1.5 wt % of the total weight of the dry powdercomposition, and the DSPE-PEG2000 is present at from about 0.15 wt % toabout 1.5 wt % of the total weight of the dry powder composition. In afurther embodiment, the DSPE-PEG2000 is present at from about 0.45 wt %to about 1.05 wt % of the total weight of the dry powder composition. Ina further embodiment, the DSPE-PEG2000 is present at about 0.75 wt % ofthe total weight of the dry powder composition. In some embodiments, thecompound of Formula (I) or (II), or a pharmaceutically acceptable saltthereof is present at about 1.5 wt % of the total weight of the drypowder composition. In some embodiments, the compound of Formula (I) or(II) is present at about 1.5 wt % of the total weight of the dry powdercomposition.

In some embodiments, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at from about 0.1 wt % to about 3 wt %, from about 0.5 wt %to about 2 wt %, from about 1 wt % to about 2 wt %, from about 1.2 wt %to about 1.8 wt %, from about 1 wt % to about 1.5 wt %, from about 1.4wt % to about 1.6 wt %, about 1 wt %, or about 1.5 wt % of the totalweight of the dry powder composition, and the weight ratio of theDSPE-PEG2000 to the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is in arange of from about 0.1:1 (DSPE-PEG2000: compound of Formula (I) or(II)) to about 1:1 (DSPE-PEG2000: compound of Formula (I) or (II)), orfrom about 0.3:1 (DSPE-PEG2000: compound of Formula (I) or (II)) toabout 0.7:1 (DSPE-PEG2000: compound of Formula (I) or (II)), e.g., about0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1.about 0.7:1, about 0.8:1, about 0.9:1, or about 1:1. In one embodiment,the weight ratio of the DSPE-PEG2000 to the compound of Formula (I) or(II), or an enantiomer, diastereomer, or a pharmaceutically acceptablesalt thereof is in a range of from about 0.1:1 (DSPE-PEG2000: compoundof Formula (I) or (II)) to about 1:1 (DSPE-PEG2000: compound of Formula(I) or (II)). In another embodiment, the weight ratio of theDSPE-PEG2000 to the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is in arange of from about 0.3:1 (DSPE-PEG2000: compound of Formula (I) or(II)) to about 0.7:1 (DSPE-PEG2000: compound of Formula (I) or (II)).

In some embodiments, the weight ratio of the DSPE-PEG2000 to thecompound of Formula (I) or (II), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof is about 0.5:1 (DSPE-PEG2000:compound of Formula (I) or (II)). At this weight ratio, in oneembodiment, the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof is presentat from about 1 wt % to about 2 wt % of the total weight of the drypowder composition, and the DSPE-PEG2000 is present at from about 0.5 wt% to about 1 wt % of the total weight of the dry powder composition. Inanother embodiment, the compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereofis present at from about 1.2 wt % to about 1.8 wt % of the total weightof the dry powder composition, and the DSPE-PEG2000 is present at fromabout 0.6 wt % to about 0.9 wt % of the total weight of the dry powdercomposition. In another embodiment, the compound of Formula (I) or (II),or an enantiomer, diastereomer, or a pharmaceutically acceptable saltthereof is present at from about 1.4 wt % to about 1.6 wt % of the totalweight of the dry powder composition, and the DSPE-PEG2000 is present atfrom about 0.7 wt % to about 0.8 wt % of the total weight of the drypowder composition. In another embodiment, the compound of Formula (I)or (II), or an enantiomer, diastereomer, or a pharmaceuticallyacceptable salt thereof is present at about 1.5 wt % of the total weightof the dry powder composition, and the DSPE-PEG2000 is present at about0.75 wt % of the total weight of the dry powder composition. In someembodiments, the compound of Formula (I) or (II), or a pharmaceuticallyacceptable salt thereof is present at each of the above-mentioned weightpercentages or weight percentage ranges in the dry powder composition.In some embodiments, the compound of Formula (I) or (II) is present ateach of the above-mentioned weight percentages or weight percentageranges in the dry powder composition.

In one embodiment, the leucine is present at from about 15 wt % to about40 wt % of the total weight of the dry powder composition. In a furtherembodiment, the leucine is present at from about 18 wt % to about 33 wt% of the total weight of the dry powder composition. In a furtherembodiment, the leucine is present at from about 20 wt % to about 30 wt%, e.g., about 20 wt %, about 25 wt %, or about 30 wt % of the totalweight of the dry powder composition. In a further embodiment, theleucine is present at from about 25 wt % to about 30 wt % of the totalweight of the dry powder composition. In a further embodiment, theleucine is present at from about 27 wt % to about 30 wt % of the totalweight of the dry powder composition. In one embodiment, the leucine ispresent at about 20 wt % of the total weight of the dry powdercomposition. In another embodiment, the leucine is present at about 30wt % of the total weight of the dry powder composition.

In some embodiments, the sugar in the dry powder composition istrehalose. In other embodiments, the sugar in the dry powder compositionis mannitol.

In one embodiment, the dry powder composition includes (a) about 1.5 wt% of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.7 wt % of the DSPE-PEG2000, (c) about 29.3 wt % of the leucine, andthe balance being (d) trehalose. In a further embodiment, (a) in the drypowder composition is about 1.5 wt % of the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1.5 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II).

In another embodiment, the dry powder composition includes (a) about 1wt % of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.5 wt % of the DSPE-PEG2000, (c) about 29.6 wt % of the leucine, andthe balance being (d) trehalose. In a further embodiment, (a) in the drypowder composition is about 1 wt % of the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II).

In another embodiment, the dry powder composition includes (a) about 1wt % of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.5 wt % of the DSPE-PEG2000, (c) about 19.7 wt % of the leucine, andthe balance being (d) trehalose. In a further embodiment, (a) in the drypowder composition is about 1 wt % of the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II).

In another embodiment, the dry powder composition includes (a) about 1.5wt % of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.7 wt % of the DSPE-PEG2000, (c) about 19.6 wt % of the leucine, andthe balance being (d) trehalose. In a further embodiment, (a) in the drypowder composition is about 1.5 wt % of the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1.5 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II).

In another embodiment, the dry powder composition includes (a) about 1.5wt % of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.7 wt % of the DSPE-PEG2000, (c) about 29.3 wt % of the leucine, andthe balance being (d) mannitol. In a further embodiment, (a) in the drypowder composition is about 1.5 wt % of the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1.5 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II).

In another embodiment, the dry powder composition includes (a) about 1.5wt % of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.75 wt % of the DSPE-PEG2000, (c) about 29.30 wt % of the leucine, and(d) about 68.45 wt % of the mannitol. In a further embodiment, (a) inthe dry powder composition is about 1.5 wt % of the compound of Formula(I) or (II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1.5 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II). In one embodiment, the dry powdercomposition includes (a) about 1.5 wt % of the compound of Formula (II),(b) about 0.75 wt % of the DSPE-PEG2000, (c) about 29.30 wt % of theleucine, and (d) about 68.45 wt % of the mannitol.

In another embodiment, the dry powder composition includes (a) about 1wt % of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.5 wt % of the DSPE-PEG2000, (c) about 29.6 wt % of the leucine, andthe balance being (d) mannitol. In a further embodiment, (a) in the drypowder composition is about 1 wt % of the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II).

In another embodiment, the dry powder composition includes (a) about 1.5wt % of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.7 wt % of the DSPE-PEG2000, (c) about 19.6 wt % of the leucine, andthe balance being (d) mannitol. In a further embodiment, (a) in the drypowder composition is about 1.5 wt % of the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1.5 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II).

In another embodiment, the dry powder composition includes (a) about 1wt % of the compound of Formula (I) or (II), or an enantiomer,diastereomer, or a pharmaceutically acceptable salt thereof, (b) about0.5 wt % of the DSPE-PEG2000, (c) about 19.7 wt % of the leucine, andthe balance being (d) mannitol. In a further embodiment, (a) in the drypowder composition is about 1 wt % of the compound of Formula (I) or(II), or a pharmaceutically acceptable salt thereof. In a furtherembodiment, (a) in the dry powder composition is about 1 wt % of thecompound of Formula (I) or (II). In some embodiments, R¹ is hexadecyl inthe compound of Formula (I). In a further embodiment, R¹ is linearhexadecyl in the compound of Formula (I), i.e., the compound of Formula(I) is a compound of Formula (II).

Mass median aerodynamic diameter (MMAD) is the value of aerodynamicdiameter for which 50% of the mass in a given aerosol is associated withparticles smaller than the median aerodynamic diameter (MAD), and 50% ofthe mass is associated with particles larger than the MAD. MMAD can bedetermined by impactor measurements, e.g., the Andersen Cascade Impactor(ACT) or the Next Generation Impactor (NGI). In some embodiments, thedry powder composition is in the form of an aerosol comprising particleswith an MMAD of from about 1 μm to about 5 μm, from about 1 μm to about3 μm, from about 1.3 μm to about 2.0 μm, or from about 1.7 μm to about2.7 μm, as measured by NGI. In one embodiment, the sugar in the drypowder composition is mannitol. In another embodiment, the sugar in thedry powder composition is trehalose.

In one embodiment, the sugar in the dry powder composition is mannitol,and the dry powder composition is in the form of an aerosol comprisingparticles with an MMAD of from about 1 μm to about 3 μm, as measured byNGI. In another embodiment, the sugar in the dry powder composition ismannitol, and the dry powder composition is in the form of an aerosolcomprising particles with an MMAD of from about 1.7 μm to about 2.7 μm,as measured by NGI.

“Fine particle fraction” or “FPF” refers to the fraction of an aerosolhaving a particle size less than 5 μm in diameter, as measured bycascade impaction. FPF is usually expressed as a percentage. FPF hasbeen demonstrated to correlate to the fraction of the powder that isdeposited in the lungs of the patient. In some embodiments, the drypowder composition is in the form of an aerosol comprising particleswith an FPF of at least 20%, at least 30%, at least 40%, at least 50%,from about 30% to about 60%, from about 35% to about 55%, or from about40% to about 50%, as measured by NGI. In one embodiment, the sugar inthe dry powder composition is mannitol. In another embodiment, the sugaris trehalose.

Tap density of a powder is the ratio of the mass of the powder to thevolume occupied by the powder after it has been tapped for a definedperiod of time. The tap density of a powder represents its random densepacking. Tap density can be determined using the method of USP BulkDensity and Tapped Density, United States Pharmacopeia convention,Rockville, Md., 10th Supplement, 4950-4951, 1999. In some embodiments,the dry powder composition comprises particles having a tap density offrom about 0.2 g/ml to about 0.8 g/ml, or from about 0.3 g/ml to about0.6 g/ml. In one embodiment, the sugar in the dry powder composition ismannitol. In another embodiment, the sugar in the dry powder compositionis trehalose.

The dry powder compositions of the present disclosure may be producedfrom liquid compositions using lyophilization or spray-dryingtechniques. When lyophilization is used, the lyophilized composition maybe milled to obtain the finely divided dry powder containing particleswithin the desired size range described above. When spray-drying isused, the process is carried out under conditions that result in afinely divided dry powder containing particles within the desired sizerange described above. Exemplary methods of preparing dry powder formsof pharmaceutical compositions are disclosed in WO 96/32149, WO97/41833, WO 98/29096, and U.S. Pat. Nos. 5,976,574, 5,985,248, and6,001,336; the disclosure of each of which is incorporated herein byreference in their entireties. Exemplary spray drying methods aredescribed in U.S. Pat. Nos. 6,848,197 and 8,197,845, the disclosure ofeach of which is incorporated herein by reference in their entireties.

In some embodiments, the dry powder compositions of the presentdisclosure are prepared by the following process. Stock solutions of acompound of Formula (I) or (II), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof and the DSPE-PEG2000 areprepared using an organic solvent, such as an alcohol (e.g.,1-propanol). Aqueous stock solutions of a sugar (e.g., mannitol ortrehalose) and leucine are also prepared. Afterwards required amounts ofthe above stock solutions are added to a mixture of water and theorganic solvent to form a spray drying feed solution. In the spraydrying feed solution, the volume ratio of water to the organic solventmay be from about 3:2 to about 1:1.

Spray drying is initiated by starting the drying gas flow and heating upthe drying gas by setting the desired inlet temperature at, for example,from about 120° C. to about 160° C., or from about 135° C. to about 150°C. After the spray dryer outlet temperature reaches a suitabletemperature, for example, at from about 55° C. to about 65° C., theliquid skid inlet is set to allow blank solvents to be atomized with theaid of nitrogen into the spray dryer, and the system is allowed to cooland stabilize. Product filter pulsing is initiated and product filterpurge flow is set, for example, to 10 to 20 scfh. After the systemstabilizes, the liquid skid inlet is switched to the feed solutionprepared above and the process is continued till the feed solution runsout. At the point when the feed solution runs out, the liquid skid inletis switched back to blank solvents which are allowed to spray for fromabout 5 to about 20 minutes. At this point, powder is collected at thebottom of the product filter. After spraying the blank solvent for fromabout 5 to about 20 minutes, the system is shut down by shutting downthe liquid lines, atomization gas, drying gas heater, drying gas inletand finally the exhaust.

In one embodiment, the dry powder compositions of the present disclosureare delivered to the lungs of a subject via inhalation using a drypowder inhaler (DPI). In one embodiment, the dry powder inhaler is asingle dose dry powder inhaler. A propellant-free device, a DPI deliversdry powder to the lungs of a subject using the subject's inspiration.The unit dose of a dry powder composition used in a DPI device is oftena dry powder blister disc of hard capsule. Exemplary DPI devicessuitable for delivering the dry powder compositions of the presentdisclosure include the devices described in the following paragraphs, aswell as the DPIs described in U.S. Pat. Nos. 6,766,799, 7,278,425 and8,496,002, the disclosure of each of which is herein incorporated byreference in their entireties.

The AIR® inhaler (Alkermes) includes a small, breath-activated systemthat delivers porous powder from a capsule. The porous particles have anaerodynamic diameter of 1-5 μm. See International Patent ApplicationPublication Nos. WO 99/66903 and WO 00/10541, the disclosure of each ofwhich is incorporated herein by reference in their entireties.

Aerolizer™ (Novartis) is a single dose dry powder inhaler. In thisdevice, dry powder medicament is stored in a capsule and released bypiercing the capsule wall with TEFLON-coated steel pins. See U.S. Pat.Nos. 6,488,027 and 3,991,761, the disclosure of each of which isincorporated herein by reference in their entireties.

Bang Olufsen provides a breath actuated inhaler using blister stripswith up to sixty doses. The dose is made available only during theinhalation by a novel trigger mechanism. The device is equipped with adose counter and can be disposed of after all doses have been used. SeeEP 1522325, the disclosure of which is incorporated herein by referencein its entirety.

Clickhaler® (Innovata PLC) is a large reservoir breath-activatedmultidose device. See U.S. Pat. No. 5,437,270, the disclosure of whichis incorporated herein by reference in its entirety.

DirectHaler™ (Direct-Haler A/S) is a single dose, pre-metered,pre-filled, disposable DPI device made from polypropylene. See U.S. Pat.No. 5,797,392, the disclosure of which is incorporated herein byreference in its entirety.

Diskus™ (GlaxoSmithKline) is a disposable small DPI device that holds upto 60 doses contained in double foil blister strips to provide moistureprotection. See GB2242134, the disclosure of which is incorporatedherein by reference in its entirety.

Eclipse™ (Aventis) is a breath actuated re-usable capsule device capableof delivering up to 20 mg of a dry power composition. The powder issucked from the capsule into a vortex chamber where a rotating ballassists in powder disaggregation as a subject inhales. See U.S. Pat. No.6,230,707 and WO 9503846, the disclosure of each of which isincorporated herein by reference in their entireties.

Flexhaler® is a plastic breath-activated dry powder inhaler and isamenable for use with the dry powder compositions provided herein.

FlowCaps® (Hovione) is a capsule-based, re-Tillable, re-usable passivedry-powder inhaler that holds up to 14 capsules. The inhaler itself ismoisture-proof. See U.S. Pat. No. 5,673,686, the disclosure of which isincorporated herein by reference in its entirety.

Gyrohaler® (Vectura) is a passive disposable DPI containing a strip ofblisters. See GB2407042, the disclosure of which is incorporated hereinby reference in its entirety.

The HandiHaler® (Boehringer Ingelheim GmbH) is a single dose DPI device.It can deliver up to 30 mg of a dry powder composition in capsules. SeeInternational Patent Application Publication No. WO 04/024156, thedisclosure of which is incorporated herein by reference in its entirety.

MicroDose DPI (Microdose Technologies) is a small electronic DPI device.It uses piezoelectric vibrator (ultrasonic frequencies) to deaggragatethe drug powder in an aluminum blister (single or multiple dose). SeeU.S. Pat. No. 6,026,809, the disclosure of which is incorporated hereinby reference in its entirety.

Nektar Dry Powder Inhaler® (Nektar) is a palm-sized and easy-to-usedevice. It provides convenient dosing from standard capsules andflow-rate-independent lung deposition.

Nektar Pulmonary Inhaler® (Nektar) efficiently removes powders from thepackaging, breaks up the particles and creates an aerosol cloud suitablefor deep lung delivery. It enables the aerosolized particles to betransported from the device to the deep lung during a patient's breath,reducing losses in the throat and upper airways. Compressed gas is usedto aerosolize the powder. See AU4090599 and U.S. Pat. No. 5,740,794, thedisclosure of each of which is incorporated herein by reference in theirentireties.

NEXT DPI™ is a device featuring multidose capabilities, moistureprotection, and dose counting. The device can be used regardless oforientation (upside down) and doses only when proper aspiratory flow isreached. See EP 1196146, U.S. Pat. No. 6,528,096, WO0178693, andWO0053158, the disclosure of each of which is incorporated herein byreference in their entireties.

Neohaler® is a capsule-based plastic breath-activated dry powderinhaler.

Oriel™ DPI is an active DPI that utilizes a piezoelectric membrane andnonlinear vibrations to aerosolize powder formulations. SeeInternational Patent Application Publication No. WO 0168169, thedisclosure of which is incorporated herein by reference in its entirety.

RS01 monodose dry powder inhaler developed by Plastiape in Italyfeatures a compact size and a simple and effective perforation systemand is suited to both gelatin and HMPC capsules.

Pressair™ is a plastic breath-activated dry powder inhaler.

Pulvinal® inhaler (Chiesi) is a breath-actuated multi-dose (100 doses)dry powder inhaler. The dry powder is stored in a reservoir which istransparent and clearly marked to indicate when the 100th dose has beendelivered. See U.S. Pat. No. 5,351,683, the disclosure of which isincorporated herein by reference in its entirety.

The Rotohaler® (GlaxoSmithKline) is a single use device that utilizescapsules. See U.S. Pat. Nos. 5,673,686 and 5,881,721, the disclosure ofeach of which is incorporated herein by reference in their entireties.

Rexam DPI (Rexam Pharma) is a single dose, reusable device designed foruse with capsules. See U.S. Pat. No. 5,651,359 and EP 0707862, thedisclosure of each of which is incorporated herein by reference in theirentireties.

S2 (Innovata PLC) is a re-useable or disposable single-dose DPI for thedelivery of a dry powder composition in high concentrations. Itsdispersion mechanism requires minimal patient effort to achieveexcellent drug delivery to the patients' lungs. S2 is easy to use andhas a passive engine so no battery or power source is required. SeeAU3320101, the disclosure of which is incorporated herein by referencein its entirety.

SkyeHaler® DPI (SkyePharma) is a multidose device containing up to 300individual doses in a single-use, or replaceable cartridge. The deviceis powered by breath and requires no coordination between breathing andactuation. See U.S. Pat. No. 6,182,655 and WO97/20589, the disclosure ofeach of which is incorporated herein by reference in their entireties.

Taifun® DPI (LAB International) is a multiple-dose (up to 200) DPIdevice. It is breath actuated and flow rate independent. The deviceincludes a unique moisture-balancing drug reservoir coupled with avolumetric dose metering system for consistent dosing. See U.S. Pat. No.6,132,394, the disclosure of which is incorporated herein by referencein its entirety.

The TurboHaler® (AstraZeneca) is described in U.S. Pat. No. 5,983,893,the disclosure of which is incorporated herein by reference in itsentirety. This DPI device is an inspiratory flow-driven, multi-dosedry-powder inhaler with a multi-dose reservoir that provides up to 200doses of a dry powder composition and a dose range from a few microgramsto 0.5 mg.

The Twisthaler® (Schering-Plough) is a multiple dose device with a dosecounting feature and is capable of 14-200 actuations. A dry powdercomposition is packaged in a cartridge that contains a desiccant. SeeU.S. Pat. No. 5,829,434, the disclosure of which is incorporated hereinby reference in its entirety.

Ultrahaler® (Aventis) combines accurate dose metering and gooddispersion. It is an easy-to-use, discrete, pocket-sized device with anumerical dose counter, dose taken indicator and a lock-out mechanism.The device is capable of delivering up to 20 mg of a dry powdercomposition. Ultrahaler® is described in U.S. Pat. No. 5,678,538 andWO2004026380, the disclosure of each of which is incorporated herein byreference in their entireties.

Xcelovair™ (Meridica/Pfizer) holds 60 pre-metered, hermetically sealeddoses in the range of 5-20 mg. The device provides moisture protectionunder accelerated conditions of 40° C./75% RH. The dispersion systemmaximizes the fine particle fraction, delivering up to 50% fine particlemass.

In another aspect, a system comprising (i) one of the dry powdercompositions described herein and (ii) a dry powder inhaler (DPI) foradministration of the dry powder composition is provided. The DPIincludes (a) a reservoir comprising the dry powder composition disclosedherein, and (b) a means for introducing the dry powder composition intothe patient via inhalation. The reservoir in one embodiment, comprisesthe dry powder composition of the present invention in a capsule or in ablister pack. The material for the shell of a capsule can be gelatin,cellulose derivatives, starch, starch derivatives, chitosan, orsynthetic plastics. The DPI may be a single dose or a multidose inhaler.In addition, the DPI may be pre-metered or device-metered. In oneembodiment, the dry powder inhaler is a single dose dry powder inhaler.

The system in one embodiment, is used for treating pulmonaryhypertension, portopulmonary hypertension, or pulmonary fibrosis. Thesystem includes the dry powder composition disclosed herein, i.e., a drypowder composition comprising a compound of Formula (I) or (II), or anenantiomer, diastereomer, or a pharmaceutically acceptable salt thereof,and a DPI. In one embodiment, the dry powder composition comprises acompound of Formula (I) or (II), or a pharmaceutically acceptable saltthereof. In another embodiment, the dry powder composition comprises acompound of Formula (I) or (II). The dry powder inhaler may be onedescribed above, may be a single dose or a multidose inhaler, and/or maybe pre-metered or device-metered. In one embodiment, the dry powderinhaler is a single dose dry powder inhaler.

In another aspect of the invention, a method for treating pulmonaryhypertension (PH) in a patient in need thereof is provided. The methodincludes administering an effective amount of the dry powder compositiondisclosed herein, i.e., a dry powder composition comprising a compoundof Formula (I) or (II), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof, to the lungs of the patient byinhalation via a dry powder inhaler. In one embodiment, the dry powdercomposition comprises a compound of Formula (I) or (II), or apharmaceutically acceptable salt thereof. In another embodiment, the drypowder composition comprises a compound of Formula (I) or (II). In oneembodiment, the administering includes aerosolizing the dry powdercomposition via a DPI to provide an aerosolized dry powder composition,and administering the aerosolized dry powder composition to the lungs ofthe patient via inhalation by the DPI. In some embodiments, theaerosolized dry powder composition comprises particles with an MMAD offrom about 1 μm to about 10 μm, from about 1 μm to about 7 μm, fromabout 1 μm to about 5 μm, or from about 1 μm to about 3 μm, as measuredby NGI. In one embodiment, the aerosolized dry powder compositioncomprises particles with an FPF of from about 40% to about 70%, fromabout 30% to about 60%, or from about 50% to about 60%, as measured byNGI.

The World Health Organization (WHO) has classified PH into five groups.Group 1 PH includes pulmonary arterial hypertension (PAH), idiopathicpulmonary arterial hypertension (IPAH), familial pulmonary arterialhypertension (FPAH), and pulmonary arterial hypertension associated withother diseases (APAH). For example, pulmonary arterial hypertensionassociated with collagen vascular disease (e.g., scleroderma),congenital shunts between the systemic and pulmonary circulation, portalhypertension and/or HIV infection are included in group 1 PH. Group 2 PHincludes pulmonary hypertension associated with left heart disease,e.g., atrial or ventricular disease, or valvular disease (e.g., mitralstenosis). WHO group 3 pulmonary hypertension is characterized aspulmonary hypertension associated with lung diseases, e.g., chronicobstructive pulmonary disease (COPD), interstitial lung disease (ILD),and/or hypoxemia. Group 4 pulmonary hypertension is pulmonaryhypertension due to chronic thrombotic and/or embolic disease. Group 4PH is also referred to as chronic thromboembolic pulmonary hypertension.Group 4 PH patients experience blocked or narrowed blood vessels due toblood clots. Group 5 PH is the “miscellaneous” category, and includes PHcaused by blood disorders (e.g., polycythemia vera, essentialthrombocythemia), systemic disorders (e.g., sarcoidosis, vasculitis)and/or metabolic disorders (e.g., thyroid disease, glycogen storagedisease).

The methods provided herein can be used to treat group 1 (i.e.,pulmonary arterial hypertension or PAH), group 2, group 3, group 4 orgroup 5 PH patients, as characterized by the WHO. In one embodiment ofthe methods, the pulmonary hypertension treated is chronicthromboembolic pulmonary hypertension.

In another embodiment of the methods, the pulmonary hypertension treatedis pulmonary arterial hypertension (PAH). In some embodiments, the PAHtreated is class I PAH, class II PAH, class III PAH, or class IV PAH, ascharacterized by the New York Heart Association (NYHA).

In one embodiment, the PAH is class I PAH, as characterized by the NYHA.

In another embodiment, the PAH is class II PAH, as characterized by theNYHA.

In yet another embodiment, the PAH is class III PAH, as characterized bythe NYHA.

In still another embodiment, the PAH is class IV PAH, as characterizedby the NYHA.

In another aspect, the present disclosure provides a method for treatingportopulmonary hypertension (PPH) in a patient in need thereof. Themethod includes administering an effective amount of the dry powdercomposition disclosed herein, i.e., a dry powder composition comprisinga compound of Formula (I) or (II), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof, to the lungs of the patient byinhalation via a dry powder inhaler. In one embodiment, the dry powdercomposition comprises a compound of Formula (I) or (II), or apharmaceutically acceptable salt thereof. In another embodiment, the drypowder composition comprises a compound of Formula (I) or (II). In oneembodiment, the administering includes aerosolizing the dry powdercomposition with a dry powder inhaler (DPI) to provide an aerosolizeddry powder composition, and administering the aerosolized dry powdercomposition to the lungs of the patient via the DPI. In someembodiments, the aerosolized dry powder composition comprises particleswith an MMAD of from about 1 μm to about 10 μm, from about 1 μm to about7 μm, from about 1 μm to about 5 μm, or from about 1 μm to about 3 μm,as measured by NGI. In one embodiment, the aerosolized dry powdercomposition comprises particles with an FPF of from about 40% to about70%, from about 30% to about 60%, or from about 50% to about 60%, asmeasured by NGI.

In some embodiments, the PH, PAH, or PPH patient treated by thedisclosed methods manifests one or more of the following therapeuticresponses: (1) a reduction in the pulmonary vascular resistance index(PVRI) from pretreatment value, (2) a reduction in mean pulmonary arterypressure from pretreatment value, (3) an increase in the hypoxemia scorefrom pretreatment value, (4) a decrease in the oxygenation index frompretreatment values, (5) improved right heart function, as compared topretreatment, and (6) improved exercise capacity (e.g., as measured bythe six-minute walk test) compared to pretreatment.

In one embodiment of the disclosed methods, the PH, PAH, or PPH patientis administered the dry powder composition once daily. In anotherembodiment of the disclosed methods, the PH, PAH, or PPH patient isadministered the dry powder composition twice daily. In still anotherembodiment of the disclosed methods, the PH, PAH, or PPH patient isadministered the dry powder composition three or more times daily. Inone embodiment, the administration is with food. In one embodiment, eachadministration comprises 1 to 5 doses (puffs) from a DPI, for example 1dose (1 puff), 2 doses (2 puffs), 3 doses (3 puffs), 4 doses (4 puffs)or 5 doses (5 puffs). The DPI, in one embodiment, is small andtransportable by the patient. In one embodiment, the dry powder inhaleris a single dose dry powder inhaler.

In still another aspect, the present disclosure provides a method fortreating pulmonary fibrosis in a patient in need thereof. The methodincludes administering an effective amount of the dry powder compositiondisclosed herein, i.e., a dry powder composition comprising a compoundof Formula (I) or (II), or an enantiomer, diastereomer, or apharmaceutically acceptable salt thereof, to the lungs of the patient byinhalation via a dry powder inhaler. In one embodiment, the dry powdercomposition comprises a compound of Formula (I) or (II), or apharmaceutically acceptable salt thereof. In another embodiment, the drypowder composition comprises a compound of Formula (I) or (II). In oneembodiment, the administering includes aerosolizing the dry powdercomposition with a DPI to form an aerosolized dry powder composition,and administering the aerosolized dry powder composition to the lungs ofthe patient via the DPI. In some embodiments, the aerosolized dry powdercomposition comprises particles with an MMAD of from about 1 μm to about10 μm, from about 1 μm to about 7 μm, from about 1 μm to about 5 μm, orfrom about 1 μm to about 3 μm, as measured by NGI. In one embodiment,the aerosolized dry powder composition comprises particles with an FPFof from about 40% to about 70%, from about 30% to about 60%, or fromabout 50% to about 60%, as measured by NGI. The patient, in oneembodiment, is administered the dry power composition once daily, twicedaily, or three or more times daily. In one embodiment, theadministration is with food. In one embodiment, each administrationcomprises 1 to 5 doses (puffs) from a DPI, for example 1 dose (1 puff),2 doses (2 puffs), 3 doses (3 puffs), 4 doses (4 puffs) or 5 doses (5puffs). The DPI, in one embodiment, is small and transportable by thepatient. In one embodiment, the dry powder inhaler is a single dose drypowder inhaler.

EXAMPLES

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments described above, are illustrative and are not to beconstrued as restricting the scope of the invention in any way.

Example 1—Preparation and Characterization of Inhalable Dry PowderFormulations Comprising the Compound of Formula (II) (TreprostinilPalmitil)

This example describes mannitol and trehalose-based dry powderformulations comprising treprostinil palmitil represented by Formula(II), their preparations by spray drying using Buchi B-290 spray dryerequipped with Inert Loop Condenser B-295 and Dehumidifier B-296, and thecharacterization and stability testing of the formulations.

The mannitol-based treprostinil palmitil dry powder formulations, withcomponents treprostinil palmitil/DSPE-PEG2000/Mannitol/Leucine(1/0.5/80/20, 1.5/0.75/80/20, 2/1/80/20, w/w) were successfully made.The feed stock was made by dissolving all components in 1-propanol/H₂Oco-solvent system (50/50, v/v), without the addition of ammoniumbicarbonate. The spray drying yields for mannitol-based dry powder wereabove 90%. The collected dry powder had spherical particles, crystallineXRD profile, and low moisture content.

The trehalose-based treprostinil palmitil dry powder formulations, withcomponents of treprostinil palmitil/DSPE-PEG2000/Trehalose/Leucine(1/0.5/80/20, 1/0.5/70/30, 1.5/0.75/80/20, 2/1/80/20, w/w), were createdby spray drying the feed stock containing all components dissolved in1-propanol/H₂O co-solvent system (50/50, v/v), without the addition ofammonium bicarbonate. The trehalose-based dry powder contained collapsedparticles, exhibiting crystalline leucine and amorphous trehalose.Trehalose-based dry powder showed good physical stability over 3 months.

Materials and Methods 1. Materials

Phosphate buffered saline: PBS, PH 7.4, Cat. No. 10010 (Lifetechnologies,), or equivalent

Sodium chloride: ACS Reagent (JT Baker, Cat. No. 3628-05), or equivalent

Treprostinil palmitil, Formula II, above

DSPE-PEG2000: N-(Methylpolyoxyethyleneoxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodiumsalt, SUNBRIGHT®DSPE-020CN (NOF, Tokyo, Japan), or equivalent

D-lactose, monohydrate, (Sigma)

L-leucine, (Sigma)

Ammonium bicarbonate (Sigma)

Absolute ethanol (Fisher Sci)

1-propanol (Fisher Sci)

2. Equipment

Buchi B-290 Spray Dryer with Inert Loop Condenser B-295, DehumidifierB-296, Two-fluid nozzle ID 0.7 mm, and high-performance cyclonicseparator (Buchi).

SEM: Zeiss-Sigma FE-SEM (Germany)

XRD: (PANalytical, Netherlands)

DSC 250, TA Instruments, New Castle, Del., USA.

Tapped density tester, JV 1000, (Copley Scientific, UK)

NGI: Next Generation Impactor, (MSP Corporation, MN, USA)

PSD: RODOS/M, (Sympatec, Germany)

Karl Fischer titrator: Aquastar, AQV33, EMD.

DLS: Möbiζ®, Atlas, (Wyatt Technology, Santa Barbara, Calif.)

High Performance Liquid Chromatograph: Waters Alliance Model 2695. HPLCsoftware: Waters Empower™ 3

Magnetic Stir Plate

3. Preparation of Dry Powder Formulation Comprising TreprostinilPalmitil, DSPE-PEG2000, Trehalose, and Leucine at a Weight Ratio of1:0.5:80:20

TABLE 1 Formulation details for trehalose-based treprostinil palmitildry powder Composition treprostinil Composition palmitil/ treprostinilDSPE-PEG2000/ palmitil/ Treh/Leu/ DSPE-PEG2000/ ammonium Trehalose/Feed- bicarbonate Leucine Solid Excipients Solvent (ABC) (mg/mL) WeightRatio (mg/ml) DSPE- 1-propanol 0.2/0.1/16/4/1 1/0.5/80/20 20.3 PEG2000/(50% in Trehalose/ water, v/v) Leucine

Preparation of Stock Solutions:

Treprostinil palmitil: 10 mg/mL in 1-propanol

DSPE-PEG2000: 10 mg/mL in 1-propanol

Trehalose: 150 mg/mL in DI water

Leucine stock: 20 mg/mL in DI water

Preparation of Spray Drying Feed Solution:

According to Table 1, the spray drying feed solution was prepared at a1:100 weight ratio of treprostinil palmitil to a total of trehalose andleucine. Final feed solution had 50% of 1-propanol and 20.3 mg/mL ofsolid (Table 2).

Trehalose and leucine stock solutions were added into water phase first,followed by the addition of 1-propanol and sonication in water bath.Then, treprostinil palmitil (Formula (II)) and DSPE-PEG2000 were addedseparately. Stirring was applied through the whole process.

TABLE 2 Preparation of spray drying stock for trehalose-based dry powderTreprostinil DSPE- 1- Palmitil PEG2000 propanol Trehalose Leucine H₂OStock 10 10 N/A 150 20 N/A (mg/mL) Weight 1 0.5 N/A 80 20 N/A ratioVolume 2 1 47 10.7 20 20.3 (100 mL) Conc 0.2 0.1 N/A 16 4 N/A (mg/mL)

Spray Drying Process of Trehalose-Based Dry Powder ContainingTreprostinil Palmitil/DSPE-PEG2000/Treh/Leu (1/0.5/80/20):

Spray drying was performed using spray dryer Buchi B-290 under thefollowing parameters: 150° C. inlet temperature, 64° C. outlettemperature, 414 L/h spray air flow (36 mm, height in rotameter), 35%m³/h as aspiration rate, and a feed-rate of 7.5 mL/min (22%). Table 3summarizes the process parameters.

TABLE 3 Parameters in spray drying process for trehalose-based drypowder Inlet T Outlet T (° C.) (° C.) Aspiration Spray gas flowFeed-rate 150 64 100% 36 mm 22% (35 m³/h) (414 L/h) (7.5 mL/min)

4. Preparation of Dry Powder Formulation Comprising TreprostinilPalmitil, DSPE-PEG2000, Mannitol, and Leucine at a Weight Ratio of1:0.5:80:20

TABLE 4 Formulation details for mannitol-based treprostinil palmitil drypowder Composition Composition treprostinil treprostinil palmitil/palmitil/ DSPE-PEG2000/ DSPE-PEG2000/ Feed- Man/Leu/ABC Man/Leu SolidExcipients Solvent (mg/mL) Weight Ratio (mg/ml) DSPE- 1-propanol0.2/0.1/16/4 1/0.5/80/20 20.3 PEG2000/ (50% in Mannitol/ water, v/v)Leucine

Preparation of Stock Solutions:

Treprostinil palmitil: 10 mg/mL in 1-propanol

DSPE-PEG2000: 10 mg/mL in 1-propanol

Mannitol: 150 mg/mL in DI water

Leucine stock: 20 mg/mL in DI water

Preparation of Spray Drying Feed Solution:

According to Table 4, the spray drying feed solution was prepared at a1:100 weight ratio of treprostinil palmitil to a total of mannitol andleucine. Final feed solution had 50% of 1-propanol and 20.3 mg/mL ofsolid (Table 5).

Mannitol and leucine stock solutions were added into water phase first,followed by the addition of propanol and sonication in water bath. Then,treprostinil palmitil and DSPE-PEG2000 were added separately. Stirringwas applied through the whole process.

TABLE 5 Preparation of spray drying stock for mannitol-based dry powderTreprostinil DSPE- 1- Palmitil PEG2000 propanol Mannitol Leucine H₂OStock 10 10 N/A 150 20 N/A (mg/mL) Weight 1 0.5 N/A 80 20 N/A RatioVolume 2 1 47 10.7 20 20.3 (100 mL) Conc 0.2 0.1 N/A 16 4 N/A (mg/mL)

Spray Drying Process of Mannitol-Based Dry Powder Composed ofTreprostinil Palmitil/DSPE-PEG2000/Mannitol/Leu (1/0.5/80/20):

Spray drying was performed using spray dryer Buchi B-290 under thefollowing parameters: 135° C. inlet temperature, 60° C. outlettemperature, 414 L/h spray air flow (36 mm, height in rotameter), 35%m³/h as aspiration rate, and a feed-rate of 7.5 mL/min (22%). Table 6summarizes the process parameters.

TABLE 6 Parameters in spray drying process for mannitol-based dry powderInlet T Outlet T Spray (° C.) (° C.) Aspiration gas flow Feed-rate 13560 100% 36 mm 22% (35 m³/h) (414 L/h) (7.5 mL/min)

5. Characterization of Dry Powder Surface Electron Microscopy (SEM)

Dry powder sample (as received) was poured on a carbon tape and thencoated with 20 nm gold (Au) using an Electron Microscopy Sciences(EMS150T ES) sputter coater. Field emission-scanning electron microscopy(FE-SEM) was used to observe the particle morphologies using aZeiss-Sigma FE-SEM (Germany) with an operating voltage of 5 keV. Theworking distance was kept between 8 to 10 mm to obtain relatively highresolution.

X-Ray Diffraction Test (XRD)

Dry powder sample (as received) was packed in the zero-background sampleholder and then X-ray diffraction (XRD) was employed for assessment ofthe structural characteristics using a PANalytical (Netherlands) X'PertDiffractometer at 45 kV and 40 mA with Cu Kα (λ=1.540598 Å) radiation ata scanning rate of 0.04 rad degrees per min. The scanning range wasbetween 4° to 40° degrees (2θ) with a time per step of 97.92 seconds anda step size of 0.0131°.

Differential Scanning Calorimetry (DSC)

Around 5-10 mg of dry powder was weighed into DSC sample pan which wasthen hermetically sealed. Test was performed as follows: equilibrate at20° C., modulate temperature 0.32° C. for 60 seconds, isothermal 1.0min, ramp 5° C./min to 180.0° C.

Particle Size Distribution (PSD) by Laser Diffraction

Around 15 mg-20 mg of dry powder was put into the required glass tube.The Sympatech-HOLOS-REDOS mode was used. Test was performed as follows:

Measuring range R1: 0.1/0.18 . . . 35 μm Trigger condition Rodosstandard trigger Disperser Rodos (RO-AS-15 mm) Disperser type RODOS/MInjector 4 mm with 0 cascade elements Primary pressure 0.5 bar

Moisture Content Test (Karl Fischer)

The moisture content in dry powder was analyzed using Karl Fischer.

Approximately 30 mg of sample was weighed out and transferred to thetitration vessel. The equipment, materials and operating parameters wereused as described below:

Materials/Equipment:

(1) Titrator: Aquastar AQV33 Karl Fischer Titrator with 5 mL buretteinstalled, with an associated balance(2) Balance: analytical balance capable of weighing up to 4-decimalplaces with an interface capable to connect to the Aquastar Titrator.

(3) Water Standard 1% NIST

(4) Dessicant 100% indicating or Molecular Sieve, Type 4A, 1/16 Pellets(5) Lint free cloth, Kimwipe(6) Weighing boats

(7) Syringe 3 mL Solutions: (1) Titrant: Aquastar CombiTitrant 2 (2)Solvent: 60/40 Methanol/Formamide Instrument Parameters and Conditions:

(1) Drift: <50 μg/min(2) Stir speed: 40%(3) Mix time: 300 sec

(4) Termination: Relative Drift Bulk and Tap Density

The density test was performed via the tapped density tester, JV 1000(Coply Scientific, UK). The following procedures were followed. Cleanglass tube, dry with compressed air; weigh glass tube, record as W1;transfer dry powder into glass tube, mark the height as A, and recordweight as W2; seal the top with parafilm; put the glass tube into a 5 mLgraduated cylinder, tapping for 10 min; mark the height as B aftertapping; remove powder from tube, clean and dry with compressed air;weight glass tube, record as W3; add water to level B, and record weightas W5; add water to level A, and record weight as W4. (assuming thedensity of water equals 1 g/mL)

Dry powder loading W2-W1 Volume of bulk density (W4-W3)/1 g/mL Volume oftap density (W5-W3)/1 g/mL Bulk density dry powder loading/volume ofbulk density Tap density dry powder loading/volume of tap density

Aerodynamic Particle Size Distribution (APSD) Using NGI

Around 20 mg of powder, filled in the size 3 Vcaps HPMC capsule, weredispersed through a commercial inhaler (Plastiape RS01) into a nextgeneration cascade impactor (NGI) (Copley Scientific, UK) operated at avolumetric flow rate of 60 L/min and actuated for 4 seconds. Drugcontent collected at each stage from the NGI apparatus was assessed byHPLC-MS. Fine particle fraction (FPF) was defined as the drug mass (<5μm) deposited in the NGI divided by the emitted dose.

HPLC Assay

Treprostinil (TRE) and treprostinil palmitil concentrations weredetermined using a Waters Alliance Model 2695 equipped with a PDADetector (Waters 2996) and Corona Charged Aerosol Detector (ThermoFisher Scientific).

-   -   Column: ACE 3 C8 HPLC Column 4.6×50 (Mac-Mod Analytical, Cat.        No. ACE1120546)    -   Column temperature: 25° C.    -   Mobile phase A: acetonitrile 25%, methanol 25%, water 50%,        formic acid 0.1%, triethylamine 0.01%    -   Mobile phase B: acetonitrile 50%, methanol 50%, formic acid        0.1%, triethylamine 0.01%    -   Flow rate: 1 mL/min    -   Gradient to measure TRE:    -   Injection volume: 50    -   UV wavelength: 270±2.4 nm    -   Samples and standards were dissolved in acetonitrile 33%,        methanol 33%, water 33%    -   Calibration was fitted by a power function Log(Area)=A        +B*Log(Conc)    -   Retention time TRE ˜1.8 min, C16TR˜7.6 min    -   Total recording time 9 min

Results 1. Batches of Mannitol-Based Treprostinil Palmitil Dry Powder

Different batches of mannitol-based treprostinil palmitil dry powderswere prepared by spray drying. In those batches, the treprostinilpalmitil amount in dry powder varied from 1 to 5% (weight ratio, w/w),while the ratio of treprostinil palmitil to DSPE-PEG2000 was kept thesame at 2 to 1. The leucine content ranged between 0 to 30% (w/w) of drypowder. The effect of ammonium bicarbonate was also investigated. Duringthe spray drying process, the inlet temperature varied from 120° C. to150° C. Table 7A shows the compositions and the inlet temperatures forthe different batches of mannitol-based treprostinil palmitil drypowders. For each batch, the amounts of treprostinil palmitil,DSPE-PEG2000, mannitol, and leucine are indicated by weight ratio. Theamounts of treprostinil palmitil and leucine are also indicated by theirapproximate weight percentages represented by their proportions in theweight ratio. Table 7B shows the targeted weight percentages oftreprostinil palmitil, DSPE-PEG2000, mannitol, and leucine in each batchcalculated based on the weight ratio.

TABLE 7A Batches of mannitol-based treprostinil palmitil dry powdersComposition treprostinil Approximate palmitil/DSPE- wt % of ApproximatePEG2000/ treprostinil wt % of Ammonium Man/Leu palmitil in leucine inbicarbonate Inlet T Batch# Weight Ratio dry powder dry powder (mg/mL) (°C.) SD-NNP-182 1/0.5/100/0 1 0 0 120 SD-NNP-181 1/0.5/100/0 1 0 0 135SD-NNP-180 1/0.5/90/10 1 10 0 135 SD-NNP-171 1/0.5/80/20 1 20 0 120SD-NNP-175 1/0.5/80/20 1 20 0 120 SD-NNP-170 1/0.5/80/20 1 20 0 135SD-NNP-179 1/0.5/80/20 1 20 0 135 SD-NNP-174 1/0.5/80/20 1 20 0 135SD-NNP-167 1/0.5/80/20 1 20 0 150 SD-NNP-168 1/0/80/20 1 20 0 150SD-NNP-172 1/0.5/80/20 1 20 0.5 120 SD-NNP-173 1/0.5/80/20 1 20 0.5 135SD-NNP-176 1/0.5/70/30 1 30 0 120 SD-NNP-177 1/0.5/70/30 1 30 0 135SD-NNP-183 1.5/0.75/80/20 1.5 20 0 135 SD-NNP-184 2/1.0/80/20 2 20 0 135SD-NNP-190 3/1.5/80/20 3 20 0 135 SD-NNP-191 5/2.5/80/20 5 20 0 135

TABLE 7B Amounts of treprostinil palmitil, DSPE-PEG2000, mannitol, andleucine expressed in weight ratios and corresponding targeted weightpercentages in batches of mannitol-based treprostinil palmitil drypowders Composition treprostinil palmitil/ DSPE- PEG2000/ Composition Wt% Man/Leu Treprostinil DSPE- Batch# Weight Ratio Palmitil PEG2000Mannitol Leucine SD-NNP-182 1/0.5/100/0 0.99 0.49 98.5 0 SD-NNP-1811/0.5/100/0 0.99 0.49 98.5 0 SD-NNP-180 1/0.5/90/10 0.99 0.49 88.7 9.85SD-NNP-171 1/0.5/80/20 0.99 0.49 78.8 19.7 SD-NNP-175 1/0.5/80/20 0.990.49 78.8 19.7 SD-NNP-170 1/0.5/80/20 0.99 0.49 78.8 19.7 SD-NNP-1791/0.5/80/20 0.99 0.49 78.8 19.7 SD-NNP-174 1/0.5/80/20 0.99 0.49 78.819.7 SD-NNP-167 1/0.5/80/20 0.99 0.49 78.8 19.7 SD-NNP-168 1/0/80/200.99 0 79.2 19.8 SD-NNP-172 1/0.5/80/20 0.99 0.49 78.8 19.7 SD-NNP-1731/0.5/80/20 0.99 0.49 78.8 19.7 SD-NNP-176 1/0.5/70/30 0.99 0.49 68.929.6 SD-NNP-177 1/0.5/70/30 0.99 0.49 68.9 29.6 SD-NNP-1831.5/0.75/80/20 1.47 0.73 78.2 19.6 SD-NNP-184 2/1.0/80/20 1.94 0.97 77.719.4 SD-NNP-190 3/1.5/80/20 2.87 1.44 76.6 19.1 SD-NNP-191 5/2.5/80/204.65 2.33 74.4 18.6

1.1. Effect of Leucine Content on the Spray Drying Recovery

The effect of leucine on the properties of mannitol-based treprostinilpalmitil dry powder was evaluated. Four leucine loads were evaluated,0%, 10%, 20% and 30%. An increase in the mannitol loads was applied tocompensate for the decrease in the leucine content.

TABLE 8 Effect of leucine on spray drying recovery and powder densityComposition treprostinil palmitil/ DSPE- Spray Leucine PEG2000/ dryingBulk Tap content, Man/Leu recovery density, density, Batch # (w/w) Wtratio (%) (g/mL) (g/mL) SD-NNP-181 0 1/0.5/100/0 33.18 N/A N/ASD-NNP-180 10 1/0.5/90/10 92.98 0.237 0.425 SD-NNP-179 20 1/0.5/80/2092.24 0.360 0.651 SD-NNP-177 30 1/0.5/70/30 85.23 0.246 0.522

Spray drying recovery (%) was low from the batch that did not containleucine. The recovery rate increased significantly as the leucine levelswere increased to 10% and 20%. The recovery rate then dropped slightlywhen the leucine content was increased further to 30% (FIG. 1). Thebatch with 20% of leucine had the highest value for powder density(Table 8).

1.2. Effect of Leucine Content (30%, 20%, 10% and 0%) on the PowderMorphology,

SEM was performed to examine the effect of leucine content on the powdersurface (FIGS. 2A-2D). The change in leucine content gave rise todifferent weight ratios of mannitol to leucine, i.e., 70/30, 80/20,90/10, and 100/0.

Treprostinil palmitil dry powder samples with different weight ratios ofmannitol to leucine (70/30, 80/20, 90/10, and 100/0) were generated. TheSEM data showed that increasing the leucine content from 20% to 30%resulted in powder with the crimped surface (FIGS. 2A and 2B). Drypowder without leucine was broken after spray drying, along with a lowrecovery rate (FIG. 2D). In further studies, 20% of leucine was used.

1.3. Effect of Leucine Content on the PSD (Tested by Laser Diffraction)

Three batches of mannitol-based dry powders, containing 10, 20 and 30%of leucine (w/w), were investigated by laser diffraction. The batchinformation was shown in Table 9.

TABLE 9 Effect of leucine on particle size distribution (laserdiffraction) Composition treprostinil palmitil/ Leucine DSPE- in dryPEG2000/ powder, Man/Leu D10, D50, D90, Batch# (w/w) Wt ratio μm μm μmSpan SD-NNP-177 30 1/0.5/70/30 1.13 4.74 9.34 1.73 SD-NNP-179 201/0.5/80/20 0.61 2.75 6.58 2.17 SD-NNP-180 10 1/0.5/90/10 0.79 3.67 8.061.98

As shown in FIG. 3, the formulation with 20% of leucine had the smallestparticle size (D50). Formulations with 20% of leucine would be used infurther studies in this example.

1.4. Effect of Various Amounts of Treprostinil Palmitil on WAD

Different amounts of treprostinil palmitil were incorporated into themannitol-based dry powder formulations, with an aim to investigate theireffects on dry powder properties. As shown in Table 10, five batches ofdry powders with treprostinil palmitil ranging from 1% to 5% and theweight ratio of treprostinil palmitil/DSPE-PEG2000 of 2:1 were prepared.

TABLE 10 Batches of mannitol-based treprostinil palmitil dry powders forstudying the effect of treprostinil palmitil content on MMADTreprostinil palmitil/ Treprostinil DSPE-PEG2000/Man/Leu Batch# Palmitil(%) (wt ratio) SD-NNP-179 1 1/0.5/80/20 SD-NNP-183 1.5 1.5/0.75/80/20SD-NNP-184 2 2/1/80/20 SD-NNP-190 3 3/1.5/80/20 SD-NNP-191 5 5/2.5/80/20

The value of MMAD was constant when there was 1 to 2% of treprostinilpalmitil in the dry powder, and it increased significantly whentreprostinil palmitil was increased to 3 to 5% (FIG. 4).

1.5. Effect of Spray Drying Inlet Temperature at 150° C., 135° C. and120° C. on Powder Morphology

Mannitol-based treprostinil palmitil dry powders with the same componentratios were generated at different inlet temperatures, i.e., 150° C.,135° C., and 120° C. (Table 11). The effect of the inlet temperature onthe dry powder morphology was investigated first. SEM revealed that lesssurface breakage was present in the dry powder samples spray dried atlower inlet temperatures of 135° C. and 120° C. (FIGS. 5A-5C). Since nosignificant difference was noticed between 135° C. and 120° C. (FIGS. 5Band 5C), the inlet temperature of 135° C. was used in furtherinvestigations.

TABLE 11 Mannitol-based dry powders spray dried at different inlettemperatures Composition treprostinil palmitil/DSPE-PEG2000/Man/LeuSpray drying inlet Batch# (Wt ratio) temperature (° C.) SD-NNP-1671/0.5/80/20 150 SD-NNP-170 1/0.5/80/20 135 SD-NNP-171 1/0.5/80/20 120

1.6. Effect of Ammonium Bicarbonate (ABC) in the Feed Stock onMannitol-Based Treprostinil Palmitil Dry Powder Morphology

The effect of ABC on the mannitol-based treprostinil palmitil dry powdermorphology was examined by adding or not ABC to the feed stock whenpreparing the dry powder (Table 12). No powder surface change wasobserved by the addition of ABC (FIGS. 6A and 6B). Therefore, ABC wouldnot be applied in mannitol-based treprostinil palmitil dry powder.

TABLE 12 Batches of mannitol-based treprostinil palmitil dry powderswith or without ammonium bicarbonate (ABC) Composition treprostinilpalmitil/DSPE-PEG2000/Man/Leu Batch # (Wt ratio) ABC (mg/ml) SD-NNP-1701/0.5/80/20 0 SD-NNP-173 1/0.5/80/20 0.5

1.7. Physical-Chemical Properties of Mannitol-Based TreprostinilPalmitil Dry Powder

The mannitol-based treprostinil palmitil dry powder was generated byspray drying a solution containing all the components. It wasanticipated that the mannitol-based dry powder would show some amorphousproperties, such as Tg in DSC test and broad peaks in powder X-raydiffraction test (XRD). FIGS. 7A and 7B showed the DSC and XRD data,respectively, from batch SD-NNP-179 which had 1% of treprostinilpalmitil and 20% of leucine. No Tg was detected and sharp peaks inpowder XRD were observed from this batch. These two characteristics werealso noticed from other batches, regardless of the difference in thecomposition and the spray drying condition.

2. Batches of Trehalose-Based Treprostinil Palmitil Dry Powder

Different batches of trehalose-based treprostinil palmitil dry powderswere made using spray drying. Table 13A shows the compositions of thebatches and the inlet temperatures used for the spray drying process.For each batch, the amounts of treprostinil palmitil, DSPE-PEG2000,trehalose, and leucine are indicated by weight ratio. Table 13B showsthe targeted weight percentages of treprostinil palmitil, DSPE-PEG2000,trehalose, and leucine in each batch calculated based on the weightratio. The batches varied in the treprostinil palmitil content from 1 to2% (weight ratio, w/w), while the ratio of treprostinil palmitil toDSPE-PEG2000 was kept the same at 2 to 1. The leucine content in thebatches was 20% or 30% (w/w). During the spray drying process, the inlettemperature varied from 110° C. to 155° C.

TABLE 13A Batches of trehalose-based treprostinil palmitil dry powdersComposition treprostinil palmitil/DSPE- PEG2000/ Organic Sugar/Leu InletT Batch Excipients solvent Wt ratio (° C.) SD-NNP-P144 Treh/Leu 1-Prop0/0/80/20 110 SD-NNP-111 Treh/Leu 1-Prop 0/0/80/20 130 SD-NNP-P143Treh/Leu 1-Prop 0/0/80/20 150 SD-NNP-169 Treh/Leu 1-Prop 1/0/80/20 150SD-NNP-112 DSPE-PEG2000/ 1-Prop 0/0.5/80/20 130 Treh/Leu SD-NNP-162DSPE-PEG2000/ 1-Prop 1/0.5/80/20 150 Treh/Leu SD-NNP-163 DSPE-PEG2000/1-Prop 1/0.5/70/30 150 Treh/Leu SD-NNP-188 DSPE-PEG2000/ 1-Prop1.5/0.75/80/20 150 Treh/Leu SD-NNP-189 DSPE-PEG2000/ 1-Prop 2/1/80/20150 Treh/Leu SD-NNP-P141 Treh/Lac/Leu EtOH 0/0/40/40/20 155 SD-NNP-140Treh/Leu EtOH 0/0/80/20 155

TABLE 13B Amounts of treprostinil palmitil, DSPE-PEG2000, trehalose, andleucine expressed in weight ratios and corresponding targeted weightpercentages in batches of trehalose-based treprostinil palmitil drypowders Composition treprostinil palmitil/ DSPE- PEG2000/ Composition wt% Sugar/Leu treprostinil DSPE- Batch Excipients (Wt ratio) palmitilPEG2000 Trehalose Leucine SD-NNP-P144 Treh/Leu 0/0/80/20 0 0 80 20SD-NNP-111 Treh/Leu 0/0/80/20 0 0 80 20 SD-NNP-P143 Treh/Leu 0/0/80/20 00 80 20 SD-NNP-169 Treh/Leu 1/0/80/20 0.99 0 79.2 19.8 SD-NNP-112 DSPE-0/0.5/80/20 0 0.50 79.6 19.9 PEG2000/ Treh/Leu SD-NNP-162 DSPE-1/0.5/80/20 0.99 0.49 78.8 19.7 PEG2000/ Treh/Leu SD-NNP-163 DSPE-1/0.5/70/30 0.99 0.49 68.9 29.6 PEG2000/ Treh/Leu SD-NNP-188 DSPE-1.5/0.75/80/20 1.47 0.73 78.2 19.6 PEG2000/ Treh/Leu SD-NNP-189 DSPE-2/1/80/20 1.94 0.97 77.7 19.4 PEG2000/ Treh/Leu SD-NNP-P141 Treh/lac/0/0/40/40/20 Not Calculated leu SD-NNP-140 Treh/leu 0/0/80/20 0 0 80 20

2.1. Effect of Leucine Content on the Powder Morphology

Trehalose-based treprostinil palmitil dry powders with two levels ofleucine (20% and 30%) were prepared (Table 14). The SEM data showed thatincreasing the leucine content from 20% to 30% resulted in the wrinkledpowder surface (FIGS. 8A and 8B).

2.2. Effect of Leucine Content on the Powder Aerosol Performance

We incorporated two levels of leucine, i.e., 20% and 30%, into thetrehalose-based treprostinil palmitil dry powders, and compared theirparticle size (D50, laser diffraction) and MMAD (Table 14).

TABLE 14 Effect of leucine content on the aerosol performance oftrehalose-based treprostinil palmitil dry powder Compositiontreprostinil palmitil/ DSPE- D50 MMAD PEG2000/ (μm), (μm), Throat +Emitted Treh/Leu PSD NGI Presep (%), dose (%), Batch Wt ratio test testNGI test NGI test SD-NNP-162 1/0.5/80/20 2.680 1.41 41.9 80.5 SD-NNP-1631/0.5/70/30 2.970 1.37 26.2 82.1

The data in Table 14 indicate that the dry powder with 30% of leucinehad a larger geometric particle size (D50) and a lower deposition ofpowder on throat and pre-separator as compared to that with 20% ofleucine. The low solubility of leucine would cause the leucine toprecipitate first. The higher amount of leucine would trigger theprecipitation quicker, generating larger particles. However, there wasno significant difference in MMAD.

2.3. Effect of Treprostinil Palmitil Content on Dry Powder AerosolPerformance.

Different amounts of treprostinil palmitil were incorporated into thetrehalose-based treprostinil palmitil dry powders, with an aim toinvestigate their effects on dry powder properties. As shown in Table15, four batches of dry powders were prepared with the treprostinilpalmitil content ranging from 1% to 2% and the weight ratio oftreprostinil palmitil/DSPE-PEG2000 fixed at 2:1.

TABLE 15 Effect of treprostinil palmitil content on dry powder aerosolperformance Composition treprostinil palmitil/ DSPE- MMAD PEG2000/ (μm),Throat + Treh/Leu NGI FPF, <5.0 μm Presep (%), Batch Wt ratio test (%),NGI test NGI test SD-NNP-162 1/0.5/80/20 1.41 40.49 41.9 SD-NNP-1631/0.5/70/30 1.37 55.93 26.2 SD-NNP-188 1.5/0.75/80/20 1.88 33.54 47.0SD-NNP-189 2/1/80/20 1.96 33.10 46.0

With more treprostinil palmitil in the dry powder, MMAD and depositionon throat and pre-separator increased while the fine particle fraction(FPF) decreased (Table 15).

2.4. Effect of Spray Drying Inlet Temperature on the Powder Morphology

The inlet temperature in spray drying process was expected to influencethe properties of dry powder, such as moisture content, particle sizeand powder morphology. Two inlet temperatures of 130° C. and 150° C.were investigated with the batches of trehalose-based vehicle drypowders shown in Table 16.

TABLE 16 Batches of trehalose-based vehicle dry powders andcorresponding inlet temperatures in spray drying process Compositiontreprostinil palmitil/ DSPE- PEG2000/ Feed stock Treh/Leu Inlet T BatchExcipients solvent Wt ratio (° C.) SD-NNP-111 Treh/Leu 1-Prop 50%0/0/80/20 130 SD-NNP-P143 Treh/Leu 1-Prop 50% 0/0/80/20 150 SD-NNP-112DSPE-PEG2000/ 1-Prop 50% 0/0.5/80/20 130 Treh/Leu

SEM revealed that a high inlet temperature of 150° C. caused the powderto break (FIGS. 9A-9C). However, to prevent high moisture in the finaldry powder, 150° C. would be used for trehalose-based dry powdercontaining treprostinil palmitil.

2.5. Physical-Chemical Properties of Trehalose-Based Dry Powder

Similar to the mannitol-based treprostinil palmitil dry powder, thetrehalose-based treprostinil palmitil dry powder was produced by spraydrying a solution containing all of the components. It was anticipatedthat the dry powder would show some amorphous properties, such as Tg inDSC and broad peaks in powder X-ray diffraction (XRD).

The batches of trehalose-based treprostinil palmitil dry powders shownin Table 17 were subjected to DSC and XRD. In the DSC test, a Tg wasobserved for all the batches, ranging from 64° C. to 80° C. The Tg couldbe increased if the powder experienced a 2^(nd) drying via overnightlyophilization, as observed in batches SD-NNP-162 and SD-NNP-163, due toa reduction of moisture in the dry powder (FIG. 10A). Compared to thatfrom the mannitol-based dry powder, the XRD from the trehalose-based drypowder showed fewer sharp peaks (FIG. 10B), which was ascribed to theamorphous state of trehalose. All of the batches showed similar XRD dataregardless of the difference in the weight ratio of the components andthe spray drying condition. Table 17 shows the additional properties ofthe batches of the dry powder, including MMAD, FPF, andthroat+pre-separator deposition.

TABLE 17 Batches of trehalose-based treprostinil palmitil dry powdersfor studying the physical-chemical properties Composition treprostinilpalmitil/ DSPE- MMAD PEG2000/ (μm), Throat + Treh/Leu NGI FPF, <5.0 μmPresep (%), Batch Wt ratio test (%), NGI test NGI test SD-NNP-1621/0.5/80/20 1.41 40.49 41.9 SD-NNP-163 1/0.5/70/30 1.37 55.93 26.2SD-NNP-188 1.5/0.75/80/20 1.88 33.54 47.0 SD-NNP-189 2/1/80/20 1.9633.10 46.0

3. Dynamic Vapor Sorption (DVS) Profile of Mannitol and Trehalose-BasedDry Powder

Moisture may be introduced into a dry powder formulation during spraydrying, packaging, and storage, causing product instability and packageissues. Upon spray drying, moisture in dry powder may be reduced viasecond drying. However, during packaging, powders may absorb moisturewhen they are exposed to the environment, even under a humidity controlcondition. Moisture absorption of the mannitol and trehalose-basedtreprostinil palmitil dry powders was examined.

As shown in FIG. 11, the mannitol-based treprostinil palmitil dry powdercould absorb up to 0.3% of moisture when the RH % was increased from 0to 40%. Compared to a lactose-based treprostinil palmitil dry powder,the mannitol-based dry powder absorbed much less moisture, probablybecause the mannitol-based dry powder contained crystalline mannitol andleucine and the stable form for both are un-hydrated form.

The moisture absorption profiles for the trehalose-based dry powders areshown in FIG. 12 (powder with 20% of leucine) and FIG. 13 (powder with30% of leucine). The weight change for the trehalose based-dry powdersreached the peak at 50% RH %. After that, the moisture uptake droppeddue to the trehalose physical form change from amorphous to crystalline.The powder formulations with 20% and 30% of leucine had similar moistureuptake data, while the latter formulation had 1% less absorption.However, the difference was significant in the desorption process. Thepowder with 30% of leucine exhibited higher moisture residual at 0% RH%.

4. Stability Test for Mannitol-Based Treprostinil Palmitil Dry Powder

The stability test for the mannitol-based treprostinil palmitil drypowders was performed at 40° C. without humidity control, over 3 months.Five batches of mannitol-based treprostinil palmitil dry powders,containing 1, 1.5, 2, 3 and 5% of treprostinil palmitil, wereinvestigated (Table 18). The NGI test showed that in general, the MMADof the dry powders rose sharply at the 2-month time point and then fellback to a level similar to that at the 1-month time point (FIG. 14). TheSEM data showed that dense small fibers grew on the powder surface(FIGS. 15A, 15B, 16A, 16B, 17A, 17B, 18A, and 18B).

TABLE 18 Batches of mannitol-based treprostinil palmitil dry powders foraccelerated stability test Treprostinil palmitil/ Treprostinil PalmitilDSPE-PEG2000/Man/Leu Batch# (%) (wt ratio) SD-NNP-179 1 1/0.5/80/20SD-NNP-183 1.5 1.5/0.75/80/20 SD-NNP-184 2 2/1/80/20 SD-NNP-190 33/1.5/80/20 SD-NNP-191 5 5/2.5/80/20

Table 19 shows the details of the stability data of the mannitol-basedtreprostinil palmitil dry powder batches. In all the batches ofmannitol-based dry powders containing 20% leucine, significant changesin MMAD and FPF were observed. Additionally, initial MMAD was lower forthe dry powders with 1, 1.5, and 2% of treprostinil palmitil, ascompared to that for the dry powder with 3 and 5% of treprostinilpalmitil.

TABLE 19 Stability data of mannitol-based treprostinil palmitil drypowder Time APSD MMAD APSD FPF PSD D50 Batch# (months) (μm) 5.0 μm (%)(μm) SD-NNP-179 0 1.76 65.8 2.75 1 3.57 35.4 3.54 2 4.83 30.7 3.70 33.99 36.3 3.32 SD-NNP-183 0 1.78 52.0 2.78 1 3.22 46.9 3.32 2 3.97 28.33.50 3 3.45 20.7 3.03 SD-NN-184 0 1.93 45.5 3.44 1 2.31 45.7 3.35 2 3.5235.8 3.88 3 2.64 51.4 3.00 SD-NNP-190 0 2.61 56.4 2.82 1 5.61 13.1 3.815 3.16 39.6 3.35 SD-NNP-191 0 2.47 56.8 2.78 1 3.92 33.1 3.99 5 3.2931.7 4.03

5. Stability Test for Trehalose-Based Dry Powder

The stability study on the trehalose-based dry powder formulations, withleucine at 20% or 30% and treprostinil palmitil ranging from 1% to 2%,was also performed under the same conditions as on the mannitol-baseddry powder formulations. Over the 3.5 months of the study period, nosignificant changes were observed (Table 20 and FIGS. 21A, 21B, 22A,22B, 23A, 23B, 24A, and 24B).

TABLE 20 Batches of trehalose-based treprostinil palmitil dry powder forstability test Treprostinil palmitil/ DSPE- PEG2000/ TreprostinilTreh/Leu 1 1.5 2 2.5 3.5 Batch# Palmitil (%) (wt ratio) month monthsmonths months months SD-NNP- 1 1/0.5/80/20 N/A Stable N/A Stable Stable162 SD-NNP- 1 1/0.5/70/30 N/A Stable N/A Stable Stable 163 SD-NNP- 1.51.5/0.75/80/20 Stable N/A Stable N/A Stable 188 SD-NNP- 2 2/1/80/20Stable N/A Stable N/A Stable 189

Table 21 shows the details of the stability data of the trahalose-basedtreprostinil palmitil dry powder batches. The MMAD increasedsignificantly for batches SD-NNP-162 and SD-NNP-188 (Table 21 and FIG.19). The FPF value decreased most notably for batch SD-NNP-162 (FIG.20). Batch SD-NNP-163 (treprostinil palmitil 1%, leucine 30%) showed thelowest and stable MMAD and the highest stable FPF.

TABLE 21 Stability data of trehalose-based treprostinil palmitil drypowders Time APSD APSD FPF PSD D50 Batch # (months) MMAD (μm) 5.0 μm (%)(μm) SD-NNP-162 0 1.41 40.5 2.68 1 1.90 37.7 2.84 2.5 2.13 38.7 2.74 3.52.80 28.1 4.56 SD-NNP-163 0 1.37 55.9 2.97 1 1.25 58.7 3.01 2.5 1.4455.5 2.90 3.5 1.39 54.6 4.62 SD-NN-188 0 1.88 33.5 2.76 1 2.41 49.7 3.002.5 2.44 56.2 4.53 3.5 2.84 51.8 2.98 SD-NNP-189 0 1.96 33.1 2.84 1 2.6736.2 3.30 2.5 2.00 47.9 4.92 3.5 2.40 42.0 3.25

Summary of Findings for Mannitol-Based Treprostinil Palmitil Dry PowderFormulations

In the mannitol-based treprostinil palmitil dry powder, addition ofleucine led to a high spray drying recovery rate. The mannitol-based drypower with 20% of leucine displayed spherical particle shape and a lowgeometric diameter (D50=2.75 μm).

In the spray drying process, the inlet temperature varied from 120° C.to 150° C. did not impact the morphology of the mannitol-basedtreprostinil palmitil dry powder. Since the moisture content was around1% from the dry powder produced under 135° C., the inlet temperature wasset at 135° C.

No glass transitions were detected from the mannitol-based treprostinilpalmitil dry powder in the DSC test, supporting the X-ray diffractiontest (XRD) result indicating crystalline materials. In addition, thespray dried mannitol-based treprostinil palmitil dry powder was lesshydroscopic, absorbing moisture less than 1% at 90% RH %.

In the stability test of the mannitol-based treprostinil palmitil drypowders, the MMAD rose sharply at 2-month time point for formulationswith 1 to 2% of treprostinil palmitil, and at 1-month time point forformulations with higher treprostinil palmitil contents. The MMADdropped at later time points. All formulations showed fibers on thepowder surface after storage.

Summary of Findings for Trehalose-Based Treprostinil Palmitil Dry PowderFormulations

The trehalose-based treprostinil palmitil dry powder containing 30%leucine had wrinkled surface and less powder deposited in the throat andpre-separator as compared to that containing 20% leucine. However, therewas no significant difference in MMAD between the two.

The inlet temperature in the spray drying process was selected at 150°C. for achieving a lower moisture content in final dry powder.

Glass transition temperature (Tg) in the range of 64 to 80° C. wasobserved, indicating an amorphous state of trehalose in the dry powder.The trehalose-based treprostinil palmitil dry powder showed higherabsorption of moisture compared to the mannitol-based treprostinilpalmitil dry powder.

In the stability test, most of the trehalose-based treprostinil palmitildry powder formulations tested exhibited no significant change in FPF.All formulations showed hair-like crystals on the powder surface afterstorage.

Taken together, the data of this example indicate that the content oftreprostinil palmitil in the range up to 2 wt % did not affect thephysical properties of the treprostinil palmitil dry powder. At 3 and 5wt % treprostinil palmitil, an increase in initial MMAD of themannitol-based powder was noted. Leucine content was found to beimportant for dry powder aerosol properties.

Example 2—Manufacture, Encapsulation, and Characterization of InhalableMannitol and Trehalose-Based Treprostinil Palmitil Dry PowderFormulations

This example describes the manufacture by spray drying and encapsulationof four treprostinil palmitil dry powder formulations, i.e.,formulations A, B, C, and D. Formulations A and D were mannitol-basedand their compositions in both weight ratios and targeted weightpercentages calculated based on the weight ratios are shown in Table 22.Formulations B and C are trehalose-based and their compositions in bothweight ratios and targeted weight percentages calculated based on theweight ratios are shown in Table 23. This example also describes thecharacterization of formulations A-D for particle size, morphology,water content, solvent content, physical state, vapor sorption profile,thermal properties, and weight loss as a function of temperature.

TABLE 22 Compositions of formulations A and D in weight ratios andtargeted weight percentages Composition treprostinil palmitil/ DSPE-PEG2000/ Composition Wt % Formu- Man/Leu Treprostinil DSPE- lation Wtratio Palmitil PEG2000 Mannitol Leucine A 1.5/0.75/80/20 1.47 0.73 78.2419.56 D 1.5/0.75/70/30 1.47 0.73 68.46 29.34

TABLE 23 Compositions of formulations B and C in weight ratios andtargeted weight percentages Composition treprostinil palmitil/ DSPE-PEG2000/ Composition Wt % Formu- Treh/Leu Treprostinil DSPE- lation Wtratio Palmitil PEG2000 Trehalose Leucine B 1.5/0.5/80/20 0.99 0.49 78.8219.70 C 1.5/0.75/70/30 1.47 0.73 68.46 29.34

1. Spray Drying Manufacture of Formulations A, B, C, and D

Treprostinil palmitil dry powder formulations A, B, C, and D weremanufactured using a BLD-200 spray dryer with in-going solids ofapproximately 55 grams each. Between each condition, a blank solventsolution was sprayed to ensure the previous formulation was cleared fromthe solution line. No additional cleaning of the spray dryer wasconducted between conditions.

Each of the four formulations was prepared as an independent solution.Solutions were prepared at room temperature without light protection.For each solution preparation, the following steps were performed:

1. Leucine was dissolved in deionized water.

2. Sugar (mannitol or trehalose) was dissolved in deionized water.

3. The aqueous solution was filtered through a 0.2 μm PVDF membrane.

4. DSPE-PEG2000 was dissolved into 1-propanol.

5. Treprostinil palmitil was dissolved into 1-propanol.

6. Organic solution was added to the stirring aqueous solution.

The spray drying formulations and process conditions are listed in Table24. Manufacturing yields ranged from 54 to 80% by mass. Packaging ofbulk dry powder of each formulation was conducted in a dry glove box.

TABLE 24 Formulations and Spray Drying Conditions Formulation A B C DComposition (wt ratio) 1.5/0.75/80/20 1/0.5/80/20 1.5/0.75/70/301.5/0.75/70/30 treprostinil treprostinil treprostinil treprostinilpalmitil/DSPE- palmitil/DSPE- palmitil/DSPE- palmitil/DSPE-PEG2000/Man./ PEG2000/Treh./ PEG2000/Treh./ PEG2000/Man./ Leu. Leu. Leu.Leu. Solvent Blend (wt %) 50/50 1-propanol/H₂O Solids Loading (wt %) 2Dryer Scale BLD-200 Chamber Pressure Negative Cyclone 3-Inch AtomizerTwo-Fluid (1650/120) Atomization Pressure 45 PSIG (217 g/min, 13 kg/hr)Drying Gas (g/min) 1300 (78 kg/hr) Feed rate (g/min) 30 Inlet Temp (°C.) 145 Outlet Temp (° C.) 60 Outlet % RH (Calc.) 8 Outlet % RS (Est.)1.3 In-going Solids (g) ~55 Solids Yield (g) 29.8 42.5 32.4 43.8 % Yield54 77 59 80

2. Powder Encapsulation

Dry power formulations were encapsulated by using an Xcelodose 600S tofill 50-51 capsules per formulation. Relative humidity of suite was lessthan 30%. A summary of the encapsulation is shown in Table 25. Forexample, capsules were made from a typical batch of formulation D(containing 1.50 wt % treprostinil palmitil, 0.75 wt % DSPE-PEG2000,68.45 wt % mannitol, and 29.30 wt % leucine) by filling in each capsule112.5 μg of treprostinil palmitil, 56.2 μg of DSPE-PEG2000, 5133.8 μg ofmannitol, and 2197.5 μg of leucine. Other batches of formulation D withwt % for each component independently varying at or within ±5% of thetypical wt % value as indicated above were observed to have equivalentproperties and performance. Capsules were collected in glass jars andheat sealed in a foil bags with 0.5 g molecular sieve desiccant.

TABLE 25 Xcelodose Performance Summary Target Mean Powder CompositionRange Machine Fill % Used Formulation (weight ratio) (mg) Yield (mg) RSD(mg) A 1.5/0.75/80/20 6.7 ± 0.7 92.4% 6.6 ± 0.2 3.3 ~370 treprostinilpalmitil/ DSPE- PEG2000/ Man./Leu. B 1/0.5/80/20 10.0 ± 1.0  96.1% 10.2± 0.3  2.5 ~530 treprostinil palmitil/ DSPE- PEG2000/ Treh./Leu. C1.5/0.75/70/30 6.7 ± 0.7 96.3% 6.6 ± 0.1 1.9 ~350 treprostinil palmitil/DSPE- PEG2000/ Treh./Leu. D 1.5/0.75/70/30 6.7 ± 0.7 91.6% 6.6 ± 0.2 2.6~370 treprostinil palmitil/ DSPE- PEG2000/ Man./Leu.

3. Analytical Characterization

Each of the four formulations was evaluated for the particle sizedistribution, particle morphology, water content, residual solvent,physical state, moisture sorption and thermal properties. A summary ofthe results is shown in Table 26.

TABLE 26 Analytical Characterization Summary Formulation A Formulation BFormulation C Formulation D Particle Size Distribution D(v 0.1), μm 0.7± 0.01 0.7 ± 0.00 0.8 ± 0.01 0.8 ± 0.03 D(v 0.5), μm 1.7 ± 0.04 1.8 ±0.02 1.8 ± 0.03 1.8 ± 0.05 D(v 0.9), μm 3.4 ± 0.06 3.6 ± 0.03 3.6 ± 0.073.5 ± 0.08 Morphology Collapsed Collapsed Collapsed Collapsed spheresspheres spheres spheres Water Content, 0.29 ± 0.01 1.65 ± 0.07 2.18 ±0.02 0.39 ± 0.03 Wt. % Residual Solvent 0.09   0.43   0.21 0.07 Wt. %Physical State Crystalline Crystalline Crystalline Crystalline leucineleucine leucine leucine Crystalline Amorphous Amorphous Crystallinemannitol trehalose trehalose mannitol Moisture Event at Event at Eventat In-Process sorption 50% RH 60% RH 60% RH Thermal Properties Tg, ° C.Not detected 83 83 Not detected Tm, ° C. 64, 164 64 64 64, 162

3.1. Particle Size Distributions

As these formulations were targeted for respiratory delivery, the targetparticle size was to be less than 5 Particle size distributions weremeasured by laser diffraction on the Malvern Mastersizer 2000 with theScirocco 2000 dry powder dispersion unit. An initial pressure titrationscreening on all samples was performed for method development (n=1), andit was observed that the results were almost identical between the threedispersive air pressures used (2.5, 3.0 and 3.5 bars) (FIG. 25). Basedon this initial screen, an additional 2 replicates (n=2) were measuredat a dispersive air pressure of 3.0 bars. Results were averaged with thedistribution shown in FIG. 26 and Table 27. For these measurements, theFraunhofer approximation model was used. A small sample tray was usedwith a feed rate of 65%, background measurement time of 10 seconds and asample measurement time of 30 seconds. Obscuration filtering was enabledto capture data between 1 to 6%.

TABLE 27 Particle size distributions of dry powder formulations A, B, C,and D Formulation D(v 0.1) μm D(v 0.5) μm D(v 0.9) μm Span A 0.7 ± 0.011.7 ± 0.04 3.4 ± 0.06 1.594 B 0.7 ± 0.00 1.8 ± 0.02 3.6 ± 0.03 1.559 C0.8 ± 0.01 1.8 ± 0.03 3.6 ± 0.07 1.538 D 0.8 ± 0.03 1.8 ± 0.05 3.5 ±0.08 1.524

3.2. Particle Morphology

Each of the four formulations was imaged at 500, 1500, and 5000magnifications using a Hitachi SU3500 scanning electron microscope.Images taken at 5000 magnification are shown in FIG. 27 (formulation A),FIG. 28 (formulation B), FIG. 29 (formulation C) and FIG. 30(formulation D). All the formulations contained full and collapsedspherical particles approximately 3 μm or less in diameter, consistentwith the laser diffraction results. Formulation C appeared to be themost corrugated while formulation A appeared to be the most “smooth”.The surface roughness may improve aerosol performance. For all theformulations, no significant crystalline surface formations or fusion ofparticles were observed.

3.3. Water Content

As the formulations were spray dried from a solvent mixture includingwater, samples were analyzed for water content using a Metrohm 874 OvenSample Processor. Three blanks and three water standards were testedfirst to determine system suitability prior to testing samples. Twentymilligram samples (n=3 replicates) were heated to 140° C. for eachformulation at a heating rate of 2.5° C./min, from a startingtemperature of 50° C. Water content for all the formulations was below3% by mass as shown in Table 28. Formulations containing trehalose (Band C) exhibited higher water content than those formulated withmannitol (A and D). The formulations containing a higher leucine content(C and D) also exhibited higher water content.

TABLE 28 Water Content Formulation Water Content (wt. %) A 0.29 ± 0.01 B1.65 ± 0.07 C 2.18 ± 0.02 D 0.39 ± 0.03

3.4. Residual Solvents

As the other component in the spray solvent was 1-propanol, the residualamounts of 1-propanol were determined using a headspace method on anAgilent 7890 gas chromatography system. All the samples had less than0.5% 1-propanol by mass (Table 29) and formulations with more leucine (Cand D) had lower residual 1-propanol as well as formulations containingmannitol (A and D).

TABLE 29 Residual Solvent 1-Propanol Content (wt. %) Formulation Wt %PPM A 0.09 900 B 0.43 4300 C 0.21 2100 D 0.07 700

3.5. Powder X-Ray Diffraction

Sample crystallinity was assessed using a Rigaku MiniFlex 600 PowderX-ray diffractometer. Samples were prepared on 0.2 mm Zero BackgroundHolder (ZBH) discs and run on the instrument from 3 to 40 2θ.Formulations A and D exhibited crystalline mannitol (likely polymorphicmixture) and leucine while formulations B and C exhibited crystallineleucine with amorphous trehalose.

Components may exhibit different diffraction intensities as neatmaterial compared with a spray dried formulation. Formulationscontaining mannitol appeared to have similar diffraction patterns.Trehalose containing formulations also appeared to have similaramorphous diffraction patterns.

3.6. Differential Scanning Calorimetry (DSC)

Thermal transitions can be used to predict stability of the formulation.The four formulations were scanned on a TA Instruments Q2000differential scanning calorimeter. Samples were equilibrated in a dryenvironment (<5% RH) overnight before they were hermetically sealed andrun on the instrument. The modulation was set to ±1.5° C./min with aheating ramp rate of 2.5° C./min from 0 to 180° C. A thermal event at64° C. was observed for all the samples, and this event may correspondwith the melt of treprostinil palmitil or DSPE-PEG2000.

No crystallization events were observed for the samples, a positiveindicator for thermal stability. No glass transitions were detected forthe mannitol-based formulations (A and D), supporting the powder X-raydiffraction (PXRD) results for crystalline material. Formulations A andD had a melt at 164° C., consistent with the melting temperature ofmannitol. Formulations B and C exhibited glass transition at 83° C.,likely due to amorphous trehalose. Formulation B also had thermal eventsat 133° C. and 158° C. not observed in other samples.

3.7. Thermal Gravimetric Analysis (TGA)

Thermal decomposition data for the formulations was measured using a TAInstruments Discovery Thermogravimetric Analyzer. Samples were run from0 to 300° C. at a rate of 2.5° C./min. The formulations began to rapidlydecompose after 180° C. Changes in weight were observed at around 100°C., corresponding with water content.

3.8. Dynamic Vapor Sorption

Water sorption and desorption profiles were measured on a SurfaceMeasurement Systems DVS Advantage 1. Samples were run from 0 to 90% RHat 25° C. with step changes of 10% RH. All four formulations appeared tohave a weight change event with an onset at around 50% or 60% RH,depending on the formulation. A second cycle was performed on thesamples to assess water sorption rates post-crystallization. None of thesamples was observed to change during the second cycle, and most samplesdid not retain water during the final desorption steps.

The sorption results from formulation D indicate a change at a higherhumidity (around 70% RH) as compared to the results from formulation A.A slight loss in mass of formulation D was observed at around 50% RHcompared with formulation A, which seemed to continually lose mass evenwith sorption of addition moisture from a higher humidity.

Uptake rate for the trehalose formulation indicates that it tookapproximately 50 to 90 minutes (depending on the level of leucinecontent) to reach moisture equilibration at a relative humidity of 40%.A lower leucine content took moisture faster than one with a higherleucine content.

3.9. Aerosol Performance of Capsules of Formulations A, C, and DAssessed by Aerodynamic Particle Size Distribution (APSD) by NGI

Capsules of treprostinil palmitil dry powder formulations A, C, and Dwere stored for 1-3 months at 25° C. or 40° C. There was no change inthe appearance of the encapsulated dry powder, e.g., no browning orobvious growth or change in hardness. Particle size distribution (PSD)of the dry powder formulations measured by laser diffraction wasunaffected after 3 months of storage at 25° C. or 40° C.

Treprostinil palmitil dry powder formulations A, C, and D wereencapsulated, and the capsules were stored for 1 month, 2 months, or 3months at 40° C., or were stored for 3 months at 25° C. The fineparticle doses (FPDs) of the formulations from the stored capsules aswell as the initial (T=0) FPDs of the formulations were measured by NGI.Treprostinil palmitil dry powder formulations A, C, and D were alsostored as bulk for 3 months at 40° C. or 25° C., and were then filledinto capsules and dosed for FPD determination on the same day. The FPDresults for formulations A, C, and D are shown in FIGS. 31, 32, and 33,respectively. These data indicate that formulation D had the leastchange in FPD (−3.7%) after stored in capsules for 3 months at 25° C.Additionally, formulation D had a −5.2% change in FPD when stored asbulk for 3 months at 25° C. and filled into capsules and dosed on thesame day. Furthermore, for each of formulations A, C, and D, storage at40° C. did not appear predictive of long term storage at 25° C. foraerosol performance as measured by FPD. Based on these data, noconditioning or pretreatment of powder or capsules to modify aerosolperformance is needed.

The emitted doses and total recovery rates of formulations A, C, and Dfrom the capsules described above were also determined, with the resultsshown in Table 30.

TABLE 30 Emitted doses (as % of loaded dose) and total recovery rates ofencapsulated treprostinil palmitil dry powder formulations A, C, and D %Emitted % Total Formulation Conditions dose recovery A T = 0 M (n = 3)73.9 92.1 T = 1 M/40° C. (n = 3) 64.2 81.8 T = 2 M/40° C. (n = 3) 64.683.0 T = 3 M/40° C. (n = 3) 66.0 85.8 T = 3 M/25° C. (n = 3) 66.6 85.4Same Day Fill 81.7 97.8 T = 3 M/40° C. (n = 3) Same Day Fill 79.0 93.6 T= 3 M/25° C. (n = 3) C T = 0 M (n = 3) 65.7 96.3 T = 1 M/40° C. (n = 3)56.5 94.2 T = 2 M/40° C. (n = 3) 46.8 91.4 T = 3 M/40° C. (n = 3) 45.8103.7 T = 3 M/25° C. (n = 3) 52.1 93.9 Same Day Fill 80.0 99.5 T = 3M/40° C. (n = 3) Same Day Fill 77.9 98.3 T = 3 M/25° C. (n = 3) D T = 0M (n = 3) 81.8 98.0 T = 1 M/40° C. (n = 3) 75.0 94.1 T = 2 M/40° C. (n =3) 72.8 92.9 T = 3 M/40° C. (n = 3) 74.6 92.4 T = 3 M/25° C. (n = 3)80.4 98.4 Same Day Fill 88.3 106.0 T = 3 M/40° C. (n = 3) Same Day Fill79.9 96.6 T = 3 M/25° C. (n = 3)

The data above indicate that formulation D displayed the least change inemitted dose after stored in capsules for 3 months at 25° C., and afterstored as bulk for 3 months at 25° C. and then filled into capsules anddosed on the same day. Taken together, based on the changes in theaerosol performance, formulation D appears stable for at least for 3months at 25° C. Additionally, the stability data up to date supports a6 months shelf life when stored at 2-8° C.

Example 3—Determination of Aerosol Performance of Trahalose-BasedTreprostinil Palmitil Dry Powder Formulation Under Accelerated StorageCondition and Lung Pharmacokinetic Profile in Rats Following Inhalationof the Dry Powder Formulation

Trahalose-based treprostinil palmitil dry powder composed oftreprostinil palmitil, DSPE-PEG2000, trehalose (Treh) and leucine (Leu)in the weight ratio of 1:0.5:70:30 was produced by spray drying using aBuchi B-290 system as described in Example 1. The dry powder was storedin sealed glass vials at 40° C. and uncontrolled ambient humidity for anaccelerated stability study. The aerosol performance of the powder wasmeasured after 1.5, 2.5, and 3.5 months of storage.

Methods and materials

1. Dynamic Vapor Sorption (DVS) Study

The moisture absorption curve was obtained using the Dynamic VaporSorption (DVS) automated gravimetric sorption system (DVS Intrinsic1Plus, Surface Measurement System, PA, USA). Approximately 20 mg ofpowder was loaded, and subjected to a cycle of sorption/desorptionisotherm (from 0% relative humidity (RH) to 90% RH to 0% RH again, inincrements of 10% RH) at 25° C. The change in powder mass (%) with RHwas determined and plotted.

2. DPI Device & Aerodynamic Particle Size Distribution (APSD) Testing

RS01 Mod.7 DPI Device (High Resistance, code 239700002AA, Plastiape,Italy) was used in this study. Mass median aerodynamic diameter (MMAD)of the trehalose-based treprostinil palmitil dry powder was measuredusing the Next Generation Impactor (NGI) at 60 L/min.

Approximately 15 mg of the treprostinil palmitil dry powder was loadedinto a Size #3 HPMC capsule (Qualicaps, Inc.). This capsule was loadedinto the DPI device and actuated to characterize the aerosol particlesize distribution (APSD). The drug amount deposited on each impactorstage and filter was analyzed by High Performance Liquid Chromatography(HPLC).

3. Nose-Only Inhalation

The treprostinil palmitil dry powder was delivered through a 12-portnose-only inhalation chamber using a dry powder dispenser (VilniusAerosol Generator (VAG), CH Technologies, USA). Approximately 1 g of thedry powder was loaded into the VAG. The VAG had a flow rate of 8 L/minat 1.0 V for a total of 20 minutes. The dry powder delivery system wasdescribed in more detail in Li et al., “Inhaled INS1009 DemonstratesLocalized Pulmonary Vasodilation,” European Respiratory Society (ERS)International Congress, 3-7 Sep. 2016, London, United Kingdom, AbstractNo: 853952 (poster PA2845), the content of which is incorporated hereinby reference in its entirety.

4. PK Sample Collection and Analysis

Rat lung tissues were harvested immediately post delivery (˜0.5 hr), 6,12, and 24 hrs after drug dosing. Lung tissue samples were analyzed fortreprostinil palmitil and treprostinil (TRE) by LC-MS/MS. Results arereported in terms of “Treprostinil Palmitil Equivalents” to account forpost-mortem hydrolysis of treprostinil palmitil.

Treprostinil Palmitil Equivalents,ng/g=[TreprostinilPalmitil,ng/g]+[TRE,ng/g]*(MM treprostinil palmitil/MM TRE)

MM: molar mass

Results 1. Dynamic Vapor Sorption (DVS)

Dynamic vapor sorption (DVS) of the treprostinil palmitil dry powderformulation is shown in FIG. 34. When the dry powder was exposed to anincrease in RH up to 50%, the absorbed moisture increased from 0% tomore than 8% and was not completely reversible with desorption,remaining at or above 5.5%. To keep moisture content at or belowapproximately 4%, the RH exposure for this powder may be controlled to<30% during manufacture.

2. Aerodynamic Particle Size Distribution (APSD)

The APSD data of the treprostinil palmitil dry powder stored for 1.5months (T=1.5M), 2.5 months (T=2.5M), and 3.5 months (T=3.5M), as wellas the initial APSD (T=0) obtained by RS01 Mod. 7 DPI (high resistance)at 60 L/min are shown in FIG. 35 and Table 31. The distributions fromall four time points were comparable.

TABLE 31 Aerodynamic particle size distribution (APSD) data oftrehalose-based treprostinil palmitil dry powder at various time pointsfollowing storage at 40° C. Treprostinil Palmitil Deposition, % ofEmitted Dose Time Point NGI Stage 0 M 1.5 M 2.5 M 3.5 M Throat + Presep31.8 32.2 32.3 35 Stage 1 3.2 2.1 3.3 2.4 Stage 2 13.5 10.1 13 11.8Stage 3 11 11.3 11.6 11.3 Stage 4 8.6 10.7 8.5 9.6 Stage 5 6.8 7.1 7 7.4Stage 6 5.6 6.9 6 5.3 Stage 7 4.7 6.4 5.2 4.5 MOC 3.8 3.9 3.9 3.7 Filter11 9.4 9 8.9

The fine particle fraction (FPF), MMAD and geometric standard deviation(GSD) values of the aerosolized dry powder after storage at 40° C. anduncontrolled ambient humidity for up to 3.5 months are summarized inTable 32. The FPF values ranged from 54.6% to 58.7%. The MMAD valuesranged from 1.25 μm to 1.44 μm. The GSD values ranged from 3.5 to 4.1.

TABLE 32 The FPF, MMAD and GSD values of aerosolized trehalose-basedtreprostinil palmitil dry powder after storage at 40° C. anduncontrolled ambient humidity for up to 3.5 months Initial (T = 0) 1.5month 2.5 months 3.5 months FPF (%) 55.9 58.7 55.5 54.6 MMAD (μm) 1.371.25 1.44 1.39 GSD 4.11 3.51 3.92 3.64

3. Rat Lung PK Results

The concentration of treprostinil palmitil equivalent (treprostinilpalmitil plus treprostinil) in the lung after inhalation of nebulizedINS1009 or the aerosolized treprostinil palmitil dry powder issummarized in Table 33 and FIG. 36. The nebulized INS1009 containedtreprostinil palmitil and the excipients squalane and DSPE-PEG2000 at amolar ratio of 45:45:10, suspended in PBS (see Corboz et al.,“Preclinical Pharmacology and Pharmacokinetics of InhaledHexadecyl-Treprostinil (C16TR), a Pulmonary Vasodilator Prodrug,” JPharmacol Exp Ther. 363:348-357 (2017), the content of which isincorporated herein by reference in its entirety). Both nebulizedINS1009 and the C16TR (treprostinil palmitil) dry powder had similarlung PK profiles following inhalation in rats. The statisticalcomparison of these two profiles is summarized in Table 34,demonstrating comparable slopes for both profiles.

TABLE 33 Concentration profile (ng/g) of treprostinil palmitilequivalent (treprostinil palmitil plus treprostinil molar equivalent,ng/g) in the rat lung after inhalation of the trehalose-basedtreprostinil palmitil dry powder or nebulized INS1009 Trehalose-basedtreprostinil palmitil Nebulized INS1009 dry powder Time Mean No. of MeanNo. (h) (ng/g) SEM Rats (ng/g) SEM of Rats 0.5 3888.5 220.1 3 3330.7253.6 6 6 1389.6 108.8 2 1582.3 186.6 4 12 690.3 95.5 3 729.1 68.8 6 24141.7 22.3 3 205.0 41.0 6 *SEM: Standard Error of the Mean

TABLE 34 Statistical analysis of the PK profile for the treprostinilpalmitil equivalent concentration in the rat lung after inhalation ofthe trehalose- based treprostinil palmitil dry powder and nebulizedINS1009 Equation: y = a + b*x Trehalose-based treprostinil palmitilFormulation Nebulized INS1009 dry powder Slope −0.064 ± 0.006 −0.055 ±0.003 Pearson's r −0.99 −1.00

Taken together, this study indicates that the aerosol particle sizedistribution of the trehalose-based treprostinil palmitil dry powderformulation is reproducible for up to 3.5 months of storage at 40° C. insealed vials and uncontrolled RH, and that the lung PK profile of thetreprostinil palmitil dry powder formulation was comparable to that ofthe nebulized INS1009.

Example 4—Pharmacokinetic Evaluations of Mannitol and Trehalose-BasedTreprostinil Palmitil Dry Powder Formulations in Rats

In this study, the lung and plasma pharmacokinetics (PK) of twodifferent treprostinil palmitil-dry powder formulations with mannitol(i.e., formulation D as described in Example 2) or trehalose (i.e.,formulation C as described in Example 2) as their major excipients wereevaluated. The compositions of formulations D and C expressed in weightratios, targeted weight percentages calculated based on the weightratios, and actual weight percentages of the components from a typicalbatch of each formulation are summarized in Tables 35A and 35B,respectively.

TABLE 35A Composition of formulation D in weight ratio, targeted weightpercentages, and actual weight percentages of components from a typicalbatch Composition Treprostinil Palmitil/DSPE- Composition Wt %PEG2000/Man/Leu Treprostinil DSPE- Wt ratio Palmitil PEG2000 MannitolLeucine Total 1.5/0.75/70/30 Targeted 1.47 0.73 68.46 29.34 100 Actual*1.50 0.75 68.45 29.30 100 *The actual wt % values shown are typical wt %values for the components in treprostinil palmitil dry powderformulation D. Batches of formulation D with wt % for each componentindependently varying at or within ±5% of the typical wt % value asshown were observed to have equivalent properties and performance.

TABLE 35B Composition of formulation C in weight ratio, targeted weightpercentages, and actual weight percentages of components from a typicalbatch Composition Treprostinil Palmitil/DSPE- Composition Wt %PEG2000/Treh/Leu Treprostinil DSPE- Wt ratio Palmitil PEG2000 TrehaloseLeucine Total 1.5/0.75/70/30 Targeted 1.47 0.73 68.46 29.34 100 Actual**1.50 0.75 67.59 30.16 100 **The actual wt % values shown are typical wt% values for the components in treprostinil palmitil dry powderformulation C. Batches of formulation C with wt % for each componentindependently varying at or within ±5% of the typical wt % value asshown were observed to have equivalent properties and performance.

Methods

Male Sprague Dawley rats (300-400 g) were exposed to aerosols of drypowder formulation D or formulation C using a 12-port nose-onlyinhalation chamber and a Vilinius Aerosol Generator (VAG). The rats wereplaced in restraining tubes that were attached to the nose-only ports inthe chamber. In two separate studies with formulation D and formulationC, 9 rats were used in each study, and 1 port was used for collection ofaerosol drug on a filter. An abbreviated study was also performed withformulation D in which 6 rats were used, and 1 port was used for thecollection of drug amount deposited on a filter.

For the drug exposures, one gram of material was placed in the VAG.Output from the VAG was established at 1 Volt (V) and the drug wasdispersed and delivered into the nose-only chamber with a bias airflowof 8 L/min. The air entered the bottom of the nose-only chamber andexited at the top. The duration of exposure was set at 20 min. A vacuumsource (0.5 L/min) was attached to the filter and the drug sampling timewas established at 5 min. The amount of treprostinil palmitil depositedon the filter was measured by HPLC and a charged aerosol detector (CAD).The delivered drug dose was calculated from the concentration of druginhaled (from filter data), the duration of exposure, respiratory minutevolume and body weight with a deposition fraction of 1.0 used for thedrug delivered at the nose, and a deposition fraction of 0.1 used forthe drug delivered to the lungs. Doses are expressed per kg body weight.

In the two primary studies with formulation D and formulation C, bloodsamples were collected at times of 0.5, 2, 4, 6, 12 and 24 hours afterdrug exposure. The blood samples were centrifuged to extract the plasma.In these studies, respiratory tissues of the larynx, trachea,carina+bronchi and lungs were collected at times of 0.5, 6, 12 and 24hours after drug exposure. In the abbreviated study with formulation D,blood samples were obtained at times of 0.5, 2, 4, 12 and 24 hours andrespiratory tissues were harvested at times of 0.5 and 24 hours afterdrug exposure. The concentrations of treprostinil (TRE) in the plasmaand treprostinil palmitil and TRE in respiratory tissues were measuredby LC-MS/MS. For all respiratory tissues, the treprostinil palmitil(C16TR) equivalent (C16TReq) concentration was derived from thetreprostinil palmitil and TRE concentrations (C16TReq=treprostinilpalmitil+[TRE×615/390.5 ng/g]), where 615 and 390.5 are the molecularweights of treprostinil palmitil and TRE, respectively. The lungtreprostinil palmitil equivalent and plasma TRE data were used to derivethe following PK parameters: lambda z (terminal elimination rateconstant), T_(1/2), T_(max), C_(max) and AUC_(0-inf) using the PK Solverprogram in Microsoft Excel.

Results

Exposure of rats to formulation D and formulation C resulted in aslightly higher total delivered dose of 78 μg/kg body weight withformulation D compared to a total delivered dose of 58 μg/kg body weightwith formulation C. The concentration of treprostinil palmitilequivalent in the lungs, measured at 0.5 hours after exposure, was alsoslightly higher with formulation D and averaged 3072 ng/g compared to1711 ng/g with formulation C.

1. Lung and Upper Airway Treprostinil Palmitil, TRE and TreprostinilPalmitil Equivalent

With both formulation D and formulation C, the highest concentrations oftreprostinil palmitil, TRE and treprostinil palmitil equivalent in thelungs occurred at 0.5 hours after exposure. There was a slowmono-exponential decline in treprostinil palmitil and TRE over 24-hourswith the concentrations of both treprostinil palmitil and TREconsistently higher at all time points with formulation D. These resultsare illustrated for treprostinil palmitil equivalent (C16TReq) in FIG.37, which shows the slow decline in lung treprostinil palmitilequivalent concentration over 24 hours with consistently higherconcentrations of treprostinil palmitil equivalent with formulation Dcompared to formulation C. FIG. 38 shows the concentration oftreprostinil palmitil (C16TR) in the lungs after inhaled formulation Dor formulation C. FIG. 39 shows the concentration of TRE in the lungsafter inhaled formulation D or formulation C.

A comparison of the derived PK parameters for lung treprostinil palmitilequivalent found no major difference between formulations D and C forlambda z, T_(1/2) and T_(max), but a 79% higher lung treprostinilpalmitil equivalent C_(max) and a 130% higher AUC_(0-24 h) forformulation D (Table 36).

TABLE 36 Pharmacokinetic parameters of lung treprostinil palmitilequivalent after inhaled Formulation D and Formulation C lambda_zT_(1/2) T_(max) C_(max) AUC_(0-24 h) AUC_(0-inf) _(—) _(obs) Compound1/h h h ng/g μg/g*h μg/g*h Formu- 0.139 4.98 0.5 3072 31.493 32.330lation D Formu- 0.130 5.35 0.5 1711 13.674 14.331 lation CAbbreviations: Lambda z, terminal elimination rate constant; T_(1/2),half-life; T_(max), time of maximal concentration; C_(max), maximalconcentration; AUC_(0-24 h), area under the concentration curve betweentime zero and 24-hours; AUC_(0-inf) _(—) _(obs), area under theconcentration curve extrapolated to infinity.

For the deposition of treprostinil palmitil in the larynx, trachea,carina+bronchi and lungs, the majority of the treprostinil palmitil (>97percent) was deposited in the lungs. This was noted with bothformulation D and formulation C. Nasal tissue was not collected in thisstudy.

2. Plasma TRE

The highest concentration of TRE in the plasma was observed at 0.5 hourswith a slow decline over 24 hours with formulation D and a more rapiddecline with formulation C. Plasma TRE concentrations were below thelevel of detection by 24 hours with formulation C (FIG. 40).

A comparison of the derived PK parameters for plasma TRE found a 34%lower lambda z and a 51% higher T½ with formulation D compared toformulation C (Table 37). The plasma TRE C_(max) was 10% higher and theAUC_(0-t) 51% higher with formulation D compared to formulation C (Table37).

TABLE 37 Pharmacokinetic parameters of plasma TRE after inhaledformulation D and formulation C lambda_z T_(1/2) T_(max) C_(max)AUC_(0-24 h) AUC_(0-inf) _(—) _(obs) Compound 1/h h h ng/mL ng/mL*hng/mL*h Formu- 0.174 3.980 0.5 0.748 4.846 4.922 lation D Formu- 0.2612.652 0.5 0.682 3.223 3.366 lation C Abbreviations: Lambda z, terminalelimination rate constant; T_(1/2), half-life; T_(max), time of maximalconcentration; C_(max), maximal concentration; AUC_(0-24 h), area underthe concentration curve between time zero and 24-hours; AUC_(0-inf) _(—)_(obs), area under the concentration curve extrapolated to infinity.

In summary, inhalation of treprostinil palmitil dry powder formulationsD and C results in low treprostinil plasma C_(max) values and sustainedlevels of treprostinil in the plasma and lungs as compared to inhalationof treprostinil. A comparison of the PK profile in rats betweenformulation D and formulation C delivered under similar conditions i.e.a VAG output of 1 V for 20 min, resulted in a 34% higher delivered dosewith formulation D that was 78 μg/kg compared to 58 μg/kg withformulation C. The concentrations of treprostinil palmitil, TRE andtreprostinil palmitil equivalent in the lungs were consistently higherwith formulation D, likely due to the higher delivered dose withformulation D, but both formulations demonstrated a slow,mono-exponential decline over 24 hours. A similar trend was observedwith the concentrations of TRE in the plasma that were higher withformulation D, and with a slow decline over 24 hours with bothformulations. Most of the TRE and treprostinil palmitil exposureoccurred in the lungs with less than 3 percent deposited in the upperrespiratory regions of the larynx, trachea, carina+bronchi.

These results with formulation D and formulation C demonstrate a PKprofile that is similar to that previously observed with nebulizedINS1009, which displays the highest concentrations of treprostinilpalmitil equivalent in the lungs and TRE in the plasma by 30 min withtheir slow mono-exponential decline over 24 hours. However, thereappears to be a significant difference in the lung treprostinil palmitilequivalent:plasma TRE C_(max) ratio between the treprostinil palmitildry powder formulations on the one hand and nebulized INS1009 on theother. Typical lung treprostinil palmitil equivalent:plasma TRE C_(max)ratio for nebulized INS1009 is ˜800, while the ratio for thetreprostinil palmitil dry powder formulations ranges from ˜1,600-13,000.See Corboz et al., J Pharmacol Exp Ther 363: 1-10 (2017), the content ofwhich is incorporated herein by reference in its entirety. Sincepulmonary vasodilation is associated more with local activity of TREwithin the lungs and less with the level of TRE in the plasma (seeChapman R W et al., Pulm. Pharmacol. Ther. 49:104-111 (2018), thecontent of which is incorporated herein by reference in its entirety),the unexpected difference in the lung treprostinil palmitilequivalent:plasma TRE C_(max) ratio indicates that administration of thetreprostinil palmitil dry powder formulations beneficially leads tolower systemic exposure to TRE and thus minimizes potential systemicadverse events, such as reductions in systemic blood pressure.

Example 5—Evaluations of Efficacy of Mannitol and Trehalose-BasedTreprostinil Palmitil Dry Powder Formulations in Hypoxia ChallengedTelemetered Rats

This example describes the in vivo efficacy evaluations of two differenttreprostinil palmitil dry powder formulations: the mannitol-basedformulation D and the trehalose-based formulation C described in Example2 and Example 4 (Tables 35A and 35B). Efficacy was determined in ratsthat were prepared with a telemetry probe implanted in the rightventricle to measure the inhibition of the increase in right ventricularpulse pressure (RVPP) that was induced by exposure to an inhaled hypoxicgas mixture.

Methods

Experiments were performed in male Sprague Dawley rats that wereimplanted with telemetry probes in the right ventricle and descendingaorta to measure RVPP and mean systemic arterial blood pressure (mSAP).These cardiovascular parameters were measured while breathing normoxicair (21% O₂/balance N2), following a 10-min exposure to hypoxic air (10%O₂/balance N₂) and returned to breathing normoxic air. In eachexperiment, the increase in RVPP due to the hypoxia challenge (Δ RVPPdue to hypoxia) was measured before drug exposure and at 1, 6, 12 and 24hours after exposure to inhaled treprostinil palmitil dry powderformulation D or formulation C. The drug formulations were given with aVilinius Aerosol Generator (VAG) at an output of 1 Volt (V) anddelivered into a 12-port nose-only inhalation chamber for 20 min. Theaerosols were dispersed from the VAG with a bias flow of 8 L/min anddelivered to the bottom of the nose-only inhalation chamber. A filterwas connected to one of the nose-only ports and attached to a vacuumflow of 0.5 L/min for 5 min. The amount of treprostinil palmitildeposited on the filter was measured by LC-MS/MS. Plasma and respiratorytissue samples of the rats were analyzed for their concentration(s) oftreprostinil palmitil and/or TRE.

Results

Exposure of rats to inhaled hypoxia increased RVPP by 10-13 mmHg overthe normoxia values. At the end of the hypoxia challenge, the RVPPimmediately decreased within a few minutes and returned to thepre-hypoxia values within 10 minutes. The drug effects were determinedby comparing the ΔRVPP due to hypoxia at various times up to 24 hoursafter drug exposure to the combined value from 2-3 determinationsobtained before drug exposure.

The experimental results are shown in FIG. 41. In FIG. 41, the valuesare the mean±SEM (n=6-8 rats for formulation D and 4 rats forformulation C). Day −1 represents baseline values before drug exposure,and Day 0 represents values post drug exposure. ΔRVPP represents theincrease in right ventricular pulse pressure. “*” denotes P<0.05compared to combined values (1, 6 and 12 hours) before drug exposure onDay −1. The results indicate that exposure to formulation D reduced theΔRVPP due to hypoxia with statistically significant (P<0.05) inhibitionobserved at 1, 6, 12 and 24 hours. Similar results were obtained withformulation C with statistically significant (P<0.05) inhibitionobserved at 1, 6, 12 and 24 hours. By 24 hours after treatment withformulation D and formulation C, the RVPP was trending back to thebaseline values observed before drug exposures. As a control, exposureto the inhaled mannitol vehicle (containing mannitol at the targetedweight percent of 69.48 wt %, leucine at the targeted weight percent of29.78 wt %, and DSPE-PEG2000 at the targeted weight percent of 0.74 wt%) did not inhibit the ΔRVPP response to hypoxia at 1, 6, 12 and 24hours after administration (data not shown). The delivered dose offormulation D was 78 μg/kg body weight (lung treprostinil palmitilequivalent concentration of 3072 ng/g) and the delivered dose offormulation C was 58 μg/kg body weight (lung treprostinil palmitilequivalent concentration of 1711 ng/g), based upon the concentration ofdrug inhaled, the duration of exposure, body weight, respiratory minutevolume and a deposition fraction of 1.0. See Alexander D. J. et al.,Inhal. Toxicol. 20:1179-1189 (2008), the content of which isincorporated herein by reference in its entirety.

In summary, efficacy evaluations in hypoxia-challenged telemetered ratsshow that the inhaled treprostinil palmitil dry powder formulations Cand D inhibited the increase in RVPP up to 24 hours after drug exposure.By 24 hours, the RVPP responses to hypoxia were trending back to thebaseline values observed before exposure to the drugs. By comparison,nebulized INS1009 at an achieved delivered dose of 76 μg/kg inhibitedthe hypoxia-induced increase in RVPP up to 12 hours with a return to thebaseline values by 24 hours. Inhaled treprostinil at the highest doseemployed (215 μg/kg) inhibited the hypoxia-induced increase in RVPP for2 hours and at lower inhaled doses (15, 53, or 110 μg/kg) for 1 hour,respectively. These results demonstrate prolonged inhibition ofhypoxia-induced increases in RVPP by the treprostinil palmitil drypowder formulations in telemetered rats.

Example 6—Assessment of Mannitol and Trehalose-Based TreprostinilPalmitil Dry Powder Formulations on Cough and Ventilation in Guinea Pigs

This example describes studies on the effects of the mannitol-basedtreprostinil palmitil dry powder formulation D and the trehalose-basedtreprostinil palmitil dry powder formulation C (described in Example 2and Example 4; see the formulation compositions in Tables 35A and 35B)to produce cough, change ventilation and change in Penh, a dimensionlessindex of altered breathing pattern typically seen duringbronchoconstriction in conscious male guinea pigs.

Methods

Experiments were performed in male Hartley guinea pigs. After a 3-dayperiod of acclimation, the guinea pigs were placed in a whole bodyplethysmograph for the measurement of ventilation (tidal volume,respiratory rate and minute volume), Penh and cough using establishedtechniques. See Corboz et al., J Pharmacol Exp Ther 363: 1-10 (2017);Chong BTY et al., J. Pharmacol. Toxicol. Methods 39, 163-168 (1998);Lomask, Exp. and Toxicol. Pathol. 57, 13-20 (2006), the content of eachof which is incorporated herein by reference in their entireties. Coughwas measured from plethysmograph recordings showing a large inspirationfollowed by a large expiration and confirmed by manual observations,video recordings and cough sounds. The ventilation, Penh and cough datawere measured during a 15-min baseline period before the exposure to thedry powder aerosol. The test articles, which included formulation D,formulation C and their respective vehicles, i.e., the mannitol vehicle(containing mannitol at the targeted weight percent of 69.48 wt %,leucine at the targeted weight percent of 29.78 wt %, and DSPE-PEG2000at the targeted weight percent of 0.74 wt %), and the trehalose vehicle(containing trehalose at the targeted weight percent of 69.48 wt %,leucine at the targeted weight percent of 29.78 wt %, and DSPE-PEG2000at the targeted weight percent of 0.74 wt %), were then delivered as drypowders for 15 min followed by a 120 min observation period after theaerosol compounds were administered. The air for the aerosol deliveryfor all steps of the experiment was supplied by an air compressor.Typical humidity of the supplied air was around 30%, as measured.Ventilation, Penh and cough were measured before, during and afterexposure to the test articles. Aerosolized test articles were producedwith a Vilnius Aerosol generator (VAG). The air flow rate through theVAG was set at 4.5 L/min to disperse the aerosol and combined with 1L/min of humidified air (30% humidification) to facilitate aerosoldelivery to the plethysmograph and minimize problems with staticadhesion. The total inflow of air was therefore 5.5 L/min. The generatoroutput from the VAG was 1, 0.75 and 0.5 Volts with each VAG output givenfor 15 min to deliver 3 different doses of the drugs, with a higher dosebeing delivered at the higher voltage. A vacuum flow of 8 L/min wasestablished at the bottom of the plethysmograph such that the air andaerosols entered the top and exited the bottom of the system. A separatevacuum source of 0.5 L/min was connected to the filter that was attachedto a port in the plethysmograph to sample the drug (treprostinilpalmitil) concentration. The filter sampling was maintained for the fullduration of the study; i.e. 135 min, but a 15 min exposure time was usedto calculate the aerosol concentration of the drug in the nose-onlychamber and the inhaled total delivered drug dose. The filter sampleswere analyzed for the treprostinil palmitil concentration. At the end ofthe study the guinea pigs were euthanized and blood (plasma), lungs,trachea, larynx and carina+bronchi were collected to measure thetreprostinil palmitil and TRE concentrations in these samples.

Results

Exposure of formulation D, formulation C, or the mannitol and trehalosevehicles, were well tolerated and did not result in any mortality.

Aerosolized formulation D generated at 1 V and administered for 15 min(inhaled total delivered dose=35.8 μg/kg body weight) produced cough in4 of 6 guinea pigs. The average number of coughs for this exposure was24±12 coughs. At a setting of 0.75 volts and administered for 15 min(inhaled total delivered dose=12.8 μg/kg body weight), cough wasobserved in 3 of 4 guinea pigs with an average cough count of 19±7coughs. At a setting of 0.5 V and administered for 15 min (inhaled totaldelivered dose=2.3 μg/kg body weight), no cough was observed in 4 of 4guinea pigs. The mannitol vehicle aerosol generated at a setting of 1 Vadministered for 15 min did not produce cough in 4 of 4 guinea pigs(Table 38). There were no consistent changes in ventilation withformulation D or the mannitol vehicle and the small increase in Penhobserved with this drug did not reach statistical significance comparedto the mannitol vehicle.

Aerosolized formulation C generated at a setting of 1 V and administeredfor 15 min (inhaled total delivered dose=10.2 μg/kg body weight)produced cough in 3 of 6 guinea pigs with an average cough count of 10±5coughs. At a setting of 0.75 V (inhaled total delivered dose=4.7 μg/kgbody weight) and 0.5 V (inhaled total delivered dose=1.5 μg/kg bodyweight) and administered for 15 min of exposure, formulation C did notcause cough in 4 of 4 guinea pigs in either group. The trehalose vehicleat a voltage of 1 V administered for 15 min did not produce cough in 4of 4 guinea pigs (Table 38). There were no consistent changes inventilation or Penh with formulation C or the trehalose vehicle.

The lung treprostinil palmitil equivalent concentration increased as afunction of the inhaled drug dose with both formulation D andformulation C, and formulation D had approximately 3-fold higher levelsof treprostinil palmitil equivalent in the lungs compared to formulationC (Table 38). There was no difference between these two formulations inthe percentage of drug deposition in the upper airway tissues of thelarynx, trachea and carina+bronchi as most of the inhaled drug wasdeposited in the lungs (data not shown). No nasal tissues were collectedin this study.

TABLE 38 Summarized data for cough, inhaled dose, treprostinil palmitilequivalent concentration in the lungs and TRE in the plasma of guineapigs exposed to formulation D or formulation C or their vehicles Lungtreprostinil Delivered palmitil Plasma VAG Cough dose equivalent TREsetting n # (μg/kg)* (ng/g)† (ng/mL)† Formulation D Vehicle 4 0 — — —1.0 volt 6 24 35.8 633 0.0782 0.75 volt 4 19 12.8 564 0.0330 0.5 volt 40 2.3 86.9 0.0308 Formulation C Vehicle 4 0 — — — 1.0 volt 6 10 10.2 2110.0398 0.75 volt 4 0 4.7 115 0.0150 0.5 volt 4 0 1.5 59.2 0.00150${\;^{*}{Delivered}\mspace{14mu} {dose}\mspace{14mu} \left( {{\mu g}\text{/}{kg}} \right)} = \frac{C\mspace{14mu} \left( {{\mu g}\text{/L}} \right) \times {RMV}\mspace{14mu} \left( {L\text{/}\min} \right) \times D\mspace{14mu} \left( \min \right)\mspace{11mu} \times {DF}}{{BW}\mspace{14mu} ({kg})}$†Samples obtained approximately 150 min after exposure to the drug.

The results from this study demonstrate that cough occurred with bothformulations and was seen at a threshold inhaled dose of 12.8 μg/kg forformulation D and 10.2 μg/kg for formulation C. These respective dosesare 10- and 8-fold higher than the threshold dose of inhaled TRE thatcauses cough in guinea pigs which is 1.23 μg/kg. After exposure toformulation D or formulation C, the first bout of coughing occurredbetween 17- and 35-minutes which is later than the timing of cough withnebulized TRE that occurs within the first 10 min of exposure.

The concentration of treprostinil palmitil equivalent in the lungs wasapproximately three times higher with formulation D (Table 38), andthere was no difference between these two formulations in the percentageof drug deposited in the upper airways of the larynx, trachea, andcarina plus bronchi as most of the drugs were deposited in the lungs(data not shown). The concentration of TRE in the plasma was between twoto three times higher with formulation D compared to formulation C(Table 38).

Example 7—Evaluation of the Effects of Treprostinil Palmitil Dry PowderFormulation in the Treatment of an 8-Week Sugen-Hypoxia (SuHx)-InducedPulmonary Arterial Hypertension Rat Model

The Sugen-Hypoxia (SuHx)-induced PAH model in rats is a well-documentedmodel. The model replicates much of the pathology seen in the clinicaldisease. In this example, the effects of treprostinil palmitil drypowder formulation D (described in Example 2 and Example 4; see theformulation composition in Table 35A) as well as inhaled treprostinil(TRE), intravenous treprostinil (TRE), and oral Selexipag, on an 8-weekSuHx-induced pulmonary arterial hypertension (PAH) model in rats,including pulmonary arterial pressure (PAP) and other cardiovascularparameters, right ventricular hypertrophy, lung and cardiachistopathology and biomarkers associated with PAH, are assessed.

Study Groups, Test Articles and Vehicles and their Administration

Male Sprague Dawley rats weighing between 200 and 250 g are randomizedinto study groups according to weight to ensure weight ranges are evenlydistributed across groups. Table 39 summarizes the study groups and thetreatment each study group receives.

TABLE 39 Treatment Group Assignment and Treatment Information Route ofTreatment Group Group Group Treatment Dosing Adminis- Starting SurgerySize # Description Dose description tration Day Day n 1 Normoxic N/A N/AN/A 22 56 8 control 2 SuHx + inhaled N/A 170 mg at Inhalation 22 56 11dry powder 1.0 volt (QD) vehicle 3 SuHx + 57 μg/kg 90 mg at Inhalation22 56 11 treprostinil 0.5 volt (QD) palmitil dry powder formulation Dlow dose 4 SuHx + 138 μg/kg 170 mg at Inhalation 22 56 11 treprostinil1.0 volt (QD) palmitil dry powder formulation D high dose 5 SuHx + N/A 6mL Inhalation 22 56 11 nebulized (QID) vehicle 6 SuHx + 110 μg/kg 6 mLof Inhalation 22 56 11 nebulized 0.5 mM (QID) TRE 7 SuHx + IV N/A N/AContinuous 22 56 11 vehicle intravenous infusion 8 SuHx + IV 810ng/kg/min 8.75 mg/mL Continuous 22 56 11 TRE (Day 22 to intravenous 39)and 10.7 infusion mg/mL (Day 40 to 56) 9 SuHx + oral N/A 10 mL/kg Oral(BID) 22 56 11 vehicle 10 SuHx + 30 mg/kg 10 mL/kg of Oral (BID) 22 5611 Selexipag 3 mg/mL

1. Group 1—Normoxic Control Group

Normoxic control group (Group 1) receives one subcutaneous injection of100% DMSO at 2 mL/kg (vehicle for sugen) and no treatment.

2. Group 2—Treatment Group with Vehicle for Treprostinil Palmitil DryPowder Formulation D

The vehicle for treprostinil palmitil dry powder formulation D has atargeted composition of 70 wt % mannitol and 30 wt % leucine. Rats inthis vehicle treatment group (Group 2) are weighed and put in the cup ofa Vilnius Aerosol Generator (VAG) (170 mg of the vehicle is loaded) andadministered in a nose-cone chamber at 1 volt, once per day until allthe material has been aerosolized. The duration of aerosolization ismeasured. In parallel with Groups 3 and 4, the vehicle treatment forthis group is conducted for 35 days.

3. Groups 3 and 4—Treatment Groups with Treprostinil Palmitil Dry PowderFormulation D

For Groups 3 and 4, treprostinil palmitil dry powder formulation D isweighed and put in the cup of a VAG (90 mg is used and generated at 0.5Volts (V) for the targeted dose of 57 μg/kg, and 170 mg is used andgenerated at 1 V for the targeted dose of 138 μg/kg). The dry powderformulation is given once per day for 35 days and, for eachadministration, the VAG is left on until all the material isaerosolized. The duration of aerosolization is measured. The Dry powderformulation is stored at 4±2° C.

4. Group 5—Treatment Group with Nebulized Vehicle for InhaledTreprostinil (TRE)

The nebulized vehicle for inhaled treprostinil (TRE) is phosphatebuffered saline (PBS). In parallel with Group 6, rats in this nebulizedvehicle treatment group (Group 5) receive nebulized PBS in a nose conechamber 4 times over a 12-hour period each day for 35 days.

5. Group 6—Treatment Group with Nebulized Treprostinil (TRE)

The nebulized treprostinil (TRE) solution contains 0.5 mM TRE in PBS ata pH of 7.4. The solution is stored at 4±2° C. and expiration date isset 7 days after preparation. The solution is used to deliver a targeteddose of 110 μg/kg TRE, and administered to rats in Group 6 by inhalation4 times over a 12-hour period each day for 35 days.

6. Group 7—Treatment Group with Vehicle for Intravenous Treprostinil ViaContinuous Infusion

The vehicle for intravenous treprostinil is an aqueous solutioncontaining 3.0 mg/mL m-Cresol, 5.3 mg/mL NaCl, and 6.3 mg/mL sodiumcitrate, with a pH of 6.0-7.2. Rats in this group (Group 7) are eachimplanted with an osmotic pump filled with the vehicle and subject tocontinuous infusion at an infusion rate as specified for Group 8 below.In parallel with Group 8, the continuous vehicle infusion for Group 7 isto last 35 days.

7. Group 8—Treatment Group with Intravenous Treprostinil (TRE) ViaContinuous Infusion

Two solutions are prepared for the intravenous administration of TRE toGroup 8. They differ only in the concentration of TRE. Specifically, thefirst solution is an aqueous solution containing 8.75 mg/mL TRE, 3.0mg/mL m-Cresol, 5.3 mg/mL NaCl, and 6.3 mg/mL sodium citrate, with a pHof 6.0-7.2. The second solution is an aqueous solution containing 10.7mg/mL TRE, 3.0 mg/mL m-Cresol, 5.3 mg/mL NaCl, and 6.3 mg/mL sodiumcitrate, with a pH of 6.0-7.2. Each rat of this group (Group 8) receivesan intravenous infusion of TRE using an implanted osmotic pump (ALZETpump). Each ALZET pump is first filled with 2 mL of the first solutionwith 8.75 mg/mL TRE, which is sufficient to achieve continuous infusionover a 28-day period (based on an infusion rate of 2.5 μL/h) for a 450 grat and achieve a targeted dose of TRE of 810 ng/kg/min. The ALZET pumpis replaced on Day 19 of the infusion (Day 40 of the whole study) andfilled with 2 mL of the second solution with 10.7 mg/mL TRE, based on anincrease in rat weight to approximately 550 g. Derivations of the TREconcentrations in the first and second solutions are shown below. Thecontinuous IV TRE infusion is to last 35 days.

Derivation of TRE concentration (8.75 mg/mL) in the first solution:

-   -   Rat weight assumed to be 450 g    -   TRE infused=810 ng/kg/min; =364.5 ng/min; =21.87 μg/h; =524.88        μg/day; =18.37 mg/35 days    -   AlZET infusion rate=2.5 μL/hr; =60 μL/day; =2100 μL/35 days;        =2.1 mL/35 days    -   TRE Concentration=18.37 mg/2.1 mL=8.75 mg/mL

Derivation of TRE concentration (10.7 mg/mL) in the second solution

-   -   Rat weight assumed to be 550 g    -   TRE infused=810 ng/kg/min; =445.5 ng/min; =26.73 μg/h; =641.52        μg/day; =22.45 mg/35 days    -   ALZET infusion rate=2.5 μL/hr; =60 μL/day; =2100 μL/35 days;        =2.1 mL/35 days    -   TRE Concentration=22.45 mg/2.1 mL=10.7 mg/mL        8. Group 9—Treatment Group with Vehicle for Selexipag Via Oral        Administration

The vehicle for Selexipag is an aqueous solution containing 0.5% (w/v)methylcellulose with a pH of 7.5-8.0. In parallel with Group 10, rats ofthis group are dosed with the vehicle by oral gavage twice a day for 35days.

9. Group 10—Treatment Group with Selexipag Via Oral Administration

A Selexipag solution containing 3.0 mg/mL Selexipag in 0.5% (w/v) methylcellulose with a pH of 7.5 is prepared. The solution is stored at roomtemperature and expiration date is set 7 days after preparation. Thesolution is administrated to this group of rats (Group 10) by oralgavage twice a day for a targeted dose of 30 mg/kg at a volume of 10mL/kg at each administration. The Selexipag treatment is to last 35days.

Study Design

Table 39 outlines the study design, the details of which are as follows.

1. Induction of PAH

The rats are randomized into the treatment groups based on their bodyweight as described above on Day 21.

On Day 0, a solution of sugen at 10 mg/mL in DMSO is prepared, and ratsfrom Groups 2 to 10 (see Table 39) receive a single subcutaneousinjection of sugen (20 mg/kg in 2 mL/kg volume) solution and returned totheir cages. Also on Day 0, rats from Group 1 receive one subcutaneousinjection of 100% DMSO at 2 mL/kg (vehicle for sugen) and returned totheir respective cages.

Rats in Groups 2-10 are placed in cages for which the controlled air isadjusted to receive a FiO₂ equivalent to 0.10 (10%) using a mixture ofnitrogen and ambient air controlled by the ventilated cage system. Theyare kept under these hypoxic conditions for 21 days. While in hypoxia,cages are cleaned and changed once a week, exposing the rats to ambientoxygen levels for less than 10 minutes. They are exposed to ambientoxygen levels from Day 22 to Day 56. Group 1 rats remain in cagesexposed to ambient oxygen (normoxic) levels for 56 days. The rats areobserved on a daily basis for any changes in their behavior and generalhealth status.

Treatment with the test articles or vehicles is administrated from Day22 to Day 56. Food and water are given ad libitum. Daily observation ofthe behavior and general health status of the rats is done. Weekly bodyweight is taken.

2. Echocardiogram

An echocardiogram monitoring of the progression of the disease iscarried out on Day 0, Day 21 (before treatment) and on surgery day (Day56) for all the rats.

3. Blood and Lung PK Sampling

Venous blood (0.5 ml, anticoagulated with EDTA) is sampled from all rats(including the normoxic and vehicle groups), at Day 23 (just before thesecond dosing), at Day 38 (just before the next dosing) and at Day 57(24 hours after the last dose). Blood is sampled from the saphenous veinfor rats with ALZET pumps, and by the jugular vein for all other rats.Whole blood is centrifuged to yield plasma, which is stored frozen at−80° C. for analysis.

4. Dry Powder Inhalation (Groups 2-4)

During the treatment period, each rat is placed into a nose-conerestraint chamber, which is connected to a 12-port nose-only inhalationchamber (CH Technologies). Treprostinil palmitil dry powder formulationD or its vehicle is delivered using a VAG. Airflow is introduced intothe VAG at a flow rate of 7 L/min and connected to the nose-onlyinhalation chamber. For Group 3 treated with a lower dose oftreprostinil palmitil dry powder formulation D, 90 mg of treprostinilpalmitil dry powder formulation D is weighed and loaded to the VAGde-agglomerator. The VAG is set to a voltage of 0.5 V, which correspondsto 0.5 mg/L powder concentration (˜7 μg/L treprostinil palmitil). ForGroup 4 treated with a higher dose of treprostinil palmitil dry powderformulation D, 170 mg of treprostinil palmitil dry powder formulation Dis weighed and loaded to the VAG de-agglomerator, and is delivered at avoltage of 1 V, which corresponds to 1.0 mg/L powder concentration(˜14.7 μg/L treprostinil palmitil). The powder aerosol outputconcentration is continuously monitored by a portable aerosol monitor(Casella MicroDust Pro). The exact delivery time is recorded. Dry powderleft inside the VAG de-agglomerator is weighed to calculate the actualamount of the test dry powder aerosolized. A glass fiber filter, whichis connected to a vacuum source at 0.5 L/min vacuum flow, is placed onone of the exposure ports at 5 min after start of aerosolization tocollect aerosol from chamber on filter for a period of 5 minutes. Allthe filter samples are kept at 4° C. until analysis.

Rats from Group 2 receive 170 mg of the vehicle for treprostinilpalmitil dry powder formulation D, administrated at 1.0 V set up.

In this study, two different inhalation towers and sets of VAGs andlasers are used, one for the dry powder vehicle and the other fortreprostinil palmitil dry powder formulation D.

After removing the remaining powder from the de-agglomerator of the VAG,all parts of the VAG are blown with dry air. The tower is blown with dryair between Group 3 (low dose) and Group 4 (high dose) dosing, andcleaned with an aqueous solution of 0.5% sodium dodecyl sulfate (SDS),tap water and distilled water after Group 4 dosing.

5. Nebulization Inhalation (Groups 5 and 6)

Treprostinil and its vehicle PBS are administered using a nebulizer anda controller (Aeroneb Pro) from Aerogen, which is manufactured todeliver a mass mean aerosol diameter (MMAD) between 2.5 to 4 μm and arange of 0.2-0.4 mL/min of flow rate. During the treatment period, eachrat is placed into a nose-cone restraint chamber, which is connected toa 12-port nose-only inhalation chamber (CH Technologies). The volume ofthe solution to be nebulized is 6 mL with airflow of 6 L/min and theconcentration of treprostinil is 0.5 mM. A glass fiber filter is placedon one of the exposure ports and connected to a vacuum source at 0.5L/min vacuum flow for a period of 5 minutes, i.e. starting at 5 minafter the start of the nebulization and ending at 10 min.

Two inhalation towers and two separate sets of nose-cones are used; onefor Group 5 receiving PBS, and one for Group 6 receiving TRE.

Cleaning of the nebulizer is performed by sequentially running anaqueous solution of 0.5% SDS, tap water and distilled water through thenebulizer and by nebulizing PBS between each use to wash out anyresidual drug from the medication cup and through the aperture plate.The nebulization tower tubing and other materials used in thenebulization process are also cleaned with the agents described aboveonce a day. Additionally, the aerosolization tower tubing and othermaterials used in the aerosolization process are cleaned with the agentsdescribed above after each chamber dosing.

6. IV Continuous Infusion (Groups 7 and 8)

Rats from Group 8 are anesthetized with isoflurane 2% and medical gradeair. An incision is made on the back of each rat to place an ALZET pumpfilled with the first solution with 8.75 mg/mL TRE. A catheter isimplanted in the jugular vein and connected to the ALZET pump. Forcontinuous infusion over a 35-days period, on day 19 of the infusion,the catheter is temporarily clamped and the ALZET pump is replaced by anew one filled with the second solution with 10.7 mg/mL TRE. Each ratfrom Group 7 is implanted with a vehicle-filled ALZET pump.

7. Oral Administration (Groups 9 and 10)

Rats from Group 10 receive the reference compound, Selexipag by oralgavage twice a day from Day 22 to Day 56 (35 days). Care is taken tomaintain a uniform suspension of Selexipag by stirring continuously,whilst doses are being drawn up into gavage syringes, filling one gavagesyringe at a time and administering that dose before filling the nextsyringe. Doses are given by oral gavage at 10 mL/kg of body weight ateach administration. Rats from Group 9 are dosed with the vehicle twicea day by oral gavage from Day 22 to Day 56 (35 days).

Surgical Instrumentation and Measurement of Hemodynamic and FunctionalParameters in Efficacy Study Rats

1. On the selected day of surgery, 24 hours after the last dosing, ratsare anaesthetized with a mixture of 2 to 2.5% isoflurane USP (AbbotLaboratories) in oxygen, and placed on a heating pad to maintain bodytemperature.

2. Rats are tracheotomized and immediately ventilated by means of apositive-pressure rodent respirator set at 10 ml/kg body weight at afrequency of 90 strokes/min.

3. A cannula connected to a pressure transducer is inserted into theleft femoral artery to measure the systemic arterial blood pressure(SAP).

4. The heart is exposed through a sternotomy and a 20GA 1.16 in Insyteis introduced into the right ventricle and rapidly hooked up to a salinefilled PE-50 catheter connected to a transducer.

5. Following a few seconds of right ventricular pressure recording, theInsyte is further advanced into the pulmonary artery to allow PAPrecording for an additional 60 seconds.

6. Hemodynamic parameters are recorded continuously for the duration ofthe procedure or until loss of PAP signal.

7. Following hemodynamic monitoring, the blood is obtained by heartpuncture for biomarker analysis (described below).

8. After collection of the blood samples, the chest cavity is furtheropened to expose the lung. The muscle over the trachea is dissected awayto remove the lungs and heart. Harvested tissues are rinsed with PBS toremove any excess of blood before being weighed.

9. The right lung is tied off and collected immediately for drugconcentration and biomarker analysis by separating the four lobes, whichare weighed, frozen into liquid nitrogen and stored at −80° C.

10. For the histology and casting of the heart, the process is asfollows; 1) Five of the 11 rats in each of Groups 2-10 are reserved toassess the histology and biochemical parameters of the heart and aretherefore treated as described in point 11 below; 2) The other 6 rats ineach of Groups 2-10 serve to determine the Fulton Index and are treatedas described in point 12. After collection of the data for the Fultonindex, the cardiac tissue is stored at −80° C. for biomarker analysis.

11. For histology, the left lung is flushed with 0.9% NaCl. The leftlung is inflated using a 10 mL syringe filled with fixative, 10% neutralbuffered formalin (NBF) with an attached blunt tip needle (23 g). Theneedle tip is inserted into the trachea, held in place with tied suturewhile another syringe is tied to the pulmonary artery. The lung isinflated gently at physiological pressure until the lung is fully,uniformly, and consistently expanded (not allowing fixative to oozethrough lung surface). This provides optimal vascular and airwayexpansion without causing excessive tissue disruption. The needle isthen removed, suture around trachea tied, and the lung immersed in 10%NBF at a 1:20 tissue to fixative ratio. The heart is rinsed in PBS andthen immersed in 10% NBF at a 1:20 tissue to fixative ratio. The tissuesare kept in formalin for 24-48 hrs. The left lung and heart are then cutand transferred in 70% ethanol.

All fixed tissues are embedded, sliced and stained. The lung sectionsare stained with Hematoxylin and Eosin (H&E) for morphologicaldeterminations or von Willebrand Factor (VWF) for endothelial cellstaining. The heart sections are stained with H&E and either with Siriusred or Trichrome staining for collagen fibers visualization andquantification.

12. As part of the Fulton index, the heart is dissected to separate theright ventricle from the left ventricle with septum, and then weighedseparately. After collection of the data for the Fulton index, thecardiac tissue is stored at −80° C. for biomarker analysis.

Acquisition and Analysis of Experimental Data

The experimental trace is analyzed by the Clampfit software from AxonInstruments.

PAP recorded continuously for at least 1 minute or until loss of signalis used to extract the mean, diastolic, and systolic pulmonary pressure.

The systemic arterial pressure (SAP) recorded continuously is used toextract the mean, diastolic and systolic arterial pressure.

At the end of the hemodynamic parameters recording, the right and leftventricle including the septum and the lung lobes are excised todetermine wet weights.

The following hemodynamic and cardiac function parameters are quantifiedwith appropriate statistical analysis.

-   -   Mean Arterial Systemic Pressure    -   Mean Arterial Pulmonary Pressure    -   Diastolic Pulmonary Pressure    -   Systolic Pulmonary Pressure    -   Systolic Right Ventricular Pressure    -   Saturation (SO₂)    -   Weight Gain    -   Lung Weight    -   Fulton's Index    -   Heart Rate    -   Pulse Pressure

Further, molecules indicative of heart biochemistry, includingbiomarkers of oxidative stress, collagen (Sircol assay) andhydroxyproline content, uric acid, natriretic peptides: BNP, NT-proBNP(biomarkers of heart muscle stress), endothelin-1 (heart failure),angiopoetin (neovascularization), von Willebrand factor (endothelialcells), interleukin-6 (biomarker of heart attack, stroke), Toll receptorC (biomarker of cardiac diseases), plasma cytokines, atrial natriureticpeptide ANP (biomarkers for stroke, coronary artery disease, myocardialinfarction and heart failure), Toponin T/I (biomarker of heartischemia), and CPK-MB (cardiac biomarker for myocardial infarction), areexamined.

Also investigated in this example are genes linked to PAH, such as bonemorphogenetic type 2 (BMPR1), BMP-9, ABCC8, TBX4, ACVRL, SMAD 4/9, KCNA5and TET2. Additionally, in heart and lung, genes such as collagen type 1alpha 1 (COL1A1), collagen type 1 alpha 2 (COL1A2), and collagen type 3alpha 1 (COL3A1) are associated with the formation and secretion ofcollagen. P4HA1 a key enzyme in collagen biosynthesis. ACTG2 is a geneassociated with myofibroblast differentiation. Changes in the expressionof those genes are investigated as well.

It is expected that treprostinil palmitil dry powder formulation D willameliorate the pathophysiology and histopathology in the pulmonary bloodvessels and heart of Su/Hx challenged rats.

While the described invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the describedinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

Patents, patent applications, patent application publications, journalarticles and protocols referenced herein are incorporated by referencein their entireties, for all purposes.

1-131. (canceled)
 132. A dry powder composition comprising: (a) fromabout 0.1 wt % to about 3 wt % of a compound of Formula (I):

or an enantiomer, diastereomer, or a pharmaceutically acceptable saltthereof, wherein R¹ is tetradecyl, pentadecyl, hexadecyl, heptadecyl, oroctadecyl, (b) from about 0.01 wt % to about 3 wt % of DSPE-PEG2000, (c)from about 10 wt % to about 50 wt % of leucine, and the balance being(d) a sugar selected from the group consisting of trehalose andmannitol, wherein the entirety of (a), (b), (c), and (d) is 100 wt %.133. The dry powder composition of claim 132, wherein R¹ is hexadecyl.134. The dry powder composition of claim 133, wherein R¹ is linearhexadecyl.
 135. The dry powder composition of claim 132, wherein theDSPE-PEG2000 is present at from about 0.03 wt % to about 2.1 wt % of thetotal weight of the dry powder composition.
 136. The dry powdercomposition of claim 132, wherein the DSPE-PEG2000 is present at fromabout 0.05 wt % to about 1.5 wt % of the total weight of the dry powdercomposition.
 137. The dry powder composition of claim 132, whichcomprises (a) about 1.5 wt % of the compound of Formula (I), (b) about0.75 wt % of the DSPE-PEG2000, (c) about 29.30 wt % of the leucine, and(d) about 68.45 wt % of the mannitol, wherein R¹ is linear hexadecyl.138. A method for treating pulmonary hypertension in a patient in needthereof, comprising administering an effective amount of the dry powdercomposition of claim 132 to the lungs of the patient by inhalation via adry powder inhaler.
 139. The method of claim 138, wherein the pulmonaryhypertension is pulmonary arterial hypertension.
 140. The method ofclaim 139, wherein the pulmonary arterial hypertension is class Ipulmonary arterial hypertension, as characterized by the New York HeartAssociation (NYHA).
 141. The method of claim 139, wherein the pulmonaryarterial hypertension is class II pulmonary arterial hypertension, ascharacterized by the NYHA.
 142. The method of claim 139, wherein thepulmonary arterial hypertension is class III pulmonary arterialhypertension, as characterized by the NYHA.
 143. The method of claim139, wherein the pulmonary arterial hypertension is class IV pulmonaryarterial hypertension, as characterized by the NYHA.
 144. The method ofclaim 138, wherein the pulmonary hypertension is group 1 pulmonaryhypertension, as characterized by the World Health Organization (WHO).145. The method of claim 138, wherein the pulmonary hypertension isgroup 2 pulmonary hypertension, as characterized by the WHO.
 146. Themethod of claim 138, wherein the pulmonary hypertension is group 3pulmonary hypertension, as characterized by the WHO.
 147. The method ofclaim 138, wherein the pulmonary hypertension is group 4 pulmonaryhypertension, as characterized by the WHO.
 148. The method of claim 138,wherein the pulmonary hypertension is group 5 pulmonary hypertension, ascharacterized by the WHO.
 149. The method of claim 138, wherein theadministering comprises aerosolizing the dry powder composition andadministering an aerosolized dry powder composition to the lungs of thepatient via inhalation, wherein the aerosolized dry powder compositioncomprises particles with an MMAD of from about 1 μm to about 3 μm, asmeasured by NGI.
 150. The method of claim 149, wherein the aerosolizeddry powder composition comprises particles with a fine particle fractionof from about 30% to about 60%, as measured by NGI.
 151. A system fortreating pulmonary hypertension, portopulmonary hypertension, orpulmonary fibrosis, comprising: the dry powder composition of claim 132,and a dry powder inhaler (DPI).